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LSU Historical Dissertations and Theses
Graduate School
1988
Cell Division Studies of Escherichia Coli:
Expression and Protein Localization of Cell
Septation Gene, FtsA.
Chong Chon Younghae
Louisiana State University and Agricultural & Mechanical College
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Younghae, Chong Chon, "Cell Division Studies of Escherichia Coli: Expression and Protein Localization of Cell Septation Gene,
FtsA." (1988). LSU Historical Dissertations and Theses. 4564.
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Cell division stu dies o f Escherichia coli: E xp ression and p rotein
lo ca lization o f cell sep tation gene, fts A
Younghae, Chong Chon, Ph.D.
The Louisiana State University and Agricultural and Mechanical Col., 1988
UMI
300 N. Zeeb Rd.
Ann Arbor, MI 48106
Cell division studies of Escherichia c o l i : Expression and
protein localization of cell septation gene, fisA.
A Dissertation
Submitted to the Graduate Faculty of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
The Department of Microbiology
by
Younghae Chong Chon
B.P., Ewha Womans University, 1976
M.S., Louisiana State University, 1984
August 1988
ACKNOWLEDGEMENTS
I would like to express my deepest appreciation to Dr. Randall
Gayda for all of the advice and support he has extended to me over the
past
few years.
His
discussionsand
insights
have
been great
inspirations and sources of motivation throughout the course of this
investigation and invaluable to the completion of this degree.
I would also like
graduate committee,
Marion Socolofsky
Dr.
to express gratitude to the members
Eric Achberger,
and Dr.
Ding Shih
Dr.
for
Michael
their
of my
Orlowski,
timely
advice
Dr.
and
education in general.
Special thanks go to Dr. Ronald Siebeling for advice and his
students for assistance in antibody preparations and to Mrs. Margaret
C. Henk for excellent technical advice in electron microscopy.
Thanks
also to the graduate students and remaining faculty and staff of the
Department of Microbiology for making my stay in Louisiana a fine
experience.
Finally,
daughter,
work.
I
would
Hyaejeong for
like
to
thank
my
husband,
Inheung,
their love, understanding and support
and
in my
TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS
........
ii
TABLE OF CONTENTS........
iii
LIST OF TABLES ................................................... vi
LIST OF FIGURES................................................. vii
ABSTRACT
.................................................... ix
INTRODUCTION ..........................*........................... 1
LITERATURE REVIEW
I. Identification of cell division genes....................... 4
II. Roles of penicillin binding proteins in
morphogenic cycle.
..........................
12
III. Periseptal annuli andpotentialdivision sites................ 16
IV. Preliminary studies on the regulation of
ftsQAZ gene expression....................................... 18
MATERIALS AND METHODS
I. Bacterial strains,plasmids and phages.................... ..21
II. Media and growth conditions...............
III. Enzymes and chemicals.............................
21
23
IV. Methods of DNA preparation
a. Large-scale plasmid DNA isolation................ ....24
b. Small-scale (rapid) plasmid isolation................ 25
V. General genetic and recombinant DNA techniques............. 26
VI. Bacterial transformations................................. 27
VII. Electrophoretic techniques................................ 28
VIII. Extraction of DNA fragments from agarose g e l s .............. 29
iii
IX. Maxicell labelling and plasmid specified proteins.......... 30
X. Plasmid constructions.................................... .31
a. In vivo mini-Mu transposon mutagenesis............... 31
b.
Construction of FtsA-LacZ overproducing
plasmid,pYC3
c.
........................ 32
Construction of promoter fusion plasmids.............33
XI. Purification of FtsA-LacZ hybrid protein................... 34
XII. Production of FtsZ-LacZ antibodies......................... 35
XIII. Western blot analysis..................................... 36
XIV. Cell fractionation procedures..............................37
XV. Isolation of membrane fractions 0MH,0ML,IM.................. 38
XVI. Immunogold labelling and electron microscopy............... 40
XVII. Preparation of cell extracts for enzyme assays..............41
XVIII. Enzyme assays
a. 0-galactosidase assay................................42
b. CAT assay........................................... 42
c. P-lactamase assay.......................
XIX. Protein assays.
43
...................................... 44
XX. Plasmolysis of bacterial cells.............................44
RESULTS
I. Isolation of FtsA-LacZ protein fusions......................45
II. Expression of FtsA-LacZ fusion polypeptide
in maxicells............................................... 46
III. Cellular localization of the FtsA-LacZ protein............. 46
IV. Overproduction and purification
of FtsA-LacZ protein
...................................... 48
iv
V. Identification of wild type FtsA protein
by Western blots........................................... 49
VI. Cellular localization of FtsA protein
A. Western blots of cell fractions.................... ..52
B. Immunogold labelling and electron microscopy
a. Wild type cells................................54
b. Plasmolyzed cells..............................55
VII. Investigation of FtsZ gene expression
A. Promoter gene fusions and
quantitation assays.............................. 56
B. Effects of FtsA, FtsQ, FtsZ, and DnaA
on ftsZ expression.....................
DISCUSSION
58
.................................................... .60
LITERATURE CITED................................................. 70
VITA
.......................................................... 133
LIST OF TABLES
Tables
Pages
1.
Bacterial strains..........................
82
2.
Plasmids and phages........................................ 83
3.
Cellular location of FtsA andFtsA-LacZ
4.
CAT/B-lactamase specificactivities
fusions.............. 84
for different
promoter gene fusions..................................... 85
5.
FtsZ effects on
promoters.................................. 86
6.
FtsQ effects on promoters.................................. 87
7.
FtsA effects on.promoters.................................. 88
8.
DnaA46 mutants effects on promters......................... 89
LIST OF FIGURES
Figures
1.
pages
Locations of coding sequences and promoters within
the ddl-envh region of the E. coli chromosome..................... 90
2.
Proposed steps in the morphogenesis of
E. coli cells.................................
3.
4.
Diagram of periseptal annuli in lkyD~cells................ 94
Schematic representation of the expression
VeCtOr pGW7
5.
6.
96
The vector pKK232-8 .................................... 98
Restriction maps of pRGC31, Mudll 1734 and
mini-Mu insertion locations...................
7.
92
100
Autoradiograph of plasmid-specified polypeptides
labelled in UV-irradiated maxicells.............................. 102
8.
Separation of membrane fraction by sucrose
equilibrium gradient............................................ 104
9.
Schematic diagram for construction of expression
plasmid, pYC3.
10.
...............................................106
SDS-PAGE profile of proteins for purification of
FtsA-LacZ protein from temperature shifted AMC290(Alac) strain
containing plasmid pYC3.............................
11.
Identification of FtsA
12.
Immunodetection of FtsA in AMC290 strain harboring pYC40 or
108
byWestern blot analysis............110
overproducing plasmid pYC3........................................ 112
Figures
13.
pages
Cellular localization of the FtsA protein by
Western blot with FtsA antibodies................................. 114
14.
Western blot with FtsA-LacZ antibodies of cell
fractions of AMC290......
15.
116
SDS-PAGE Coomassie blue stain gel profile of AMC290 cellular
fractions analysed in immunoblot of Fig. 15....................... 118
16.
Western blot of FtsA antibodiespreabsorbed with total
membrane of cell fractions of AMC290............................. 120
17.
Electron microscopic localization of FtsA in E, coli
cells by immunogold labelling.................................... 122
18.
Phase contrast light and electron micrographs of
plasmolyzed ftsZ84(ts) filaments................................. 125
19.
Immunoelectron micrographic localization of FtsA
in ftsZ84 filament............................................... 127
20.
Localization of FtsA in plasmolyzed ftsZ84
filament by immunoelectron micrograph......................
21.
129
Promoter fusions of the ftsk-ftsZ regulatory region
to the chloramphenicol acetylase gene(Cmr).........................131
ABSTRACT
FtsA is a gene essential
for cell septation.
It has been
postulated to have both regulatory and structural functions.
To study
the structural involvement of the FtsA protein in septum formation,
four
FtsA-LacZ
protein
transposon (MudII1734).
fusions
were
constructed
using
a
mini-Mu
When maxicells containing either ftsA-lacZ or
ftsA specifying plasmids were radioactively labelled and fractionated,
FtsA-LacZ fusion protein and FtsA were located in both the membrane
and cytoplasmic fractions.
located
in
dividing
galactosidase
The FtsA-LacZ fusion proteins were also
wild
activity.
type
Cell
dividing
envelope
cells
fractions
by
measuring
from
a
fJ-
sucrose
equilibrium gradient had P-galactosidase activity in outer membraneheavy(OM„)
and outer membrane-1ight(0ML ) fractions.
To verify the
cellular location of native FtsA protein in membrane fractions,
an
expression plasmid was constructed by placing a ftsA-lacZ gene fusion
downstream from a X PL promoter.
overproduced
and
chromatography.
purified
by
The FtsA-LacZ fusion protein was
0-galactosidase
affinity
column
Polyclonal antibodies to this protein were isolated
and demonstrated to be specific for the FtsA-LacZ fusion, FtsA protein
and B-galactosidase.
Cross reactivity to FtsA protein was found in
outer membrane fractions, 0M„ and 0ML , and inner membrane fraction of
wild type cells.
These data suggest that the FtsA protein may be
functioning as an inner-outer membrane protein in septation.
IX
Electron microscopy of thin sections treated with FtsA-LacZ
antibodies and immunogold labelling also suggest that the FtsA protein
is located in the cell envelope.
of
FtsA
filamentous
protein
cells
by
Furthermore, direct identification
immuno-electron-microscopy
indicates
an
enrichment
septation sites.
X
of
in
FtsA
normal ' and
protein
near
INTRODUCTION
Cell
process
division
that
in
involves
Escherichia
the
coli
coordination
physiological growth processes including
mass and volume,
is
and
a
biological
regulation
of
several
increases in cell
length,
initiation of DNA replication,
replicated chromosomes.
complex
and segregation of
The final steps in cell division are the
coordinated invagination of the three envelope layers(inner membrane,
murein,
outer
membrane)
at
the
cell
center
(septation)
and
then
physical separation of the newly formed daughter cells.
Progress has been made in understanding cell division at the
genetic level. Conditional lethal mutants that cannot form septa have
been isolated and referred to as filamentation temperature-sensitive
(fts) mutants.
septum are
map.
A number of genes involved in the formation of the
clustered at the 2 minute region on the E. coli genetic
Based on phenotypic properties, these genes have been postulated
to have essential roles in the initiation of septum formation (ftsl
and ftsQ) and later invagination stages (ftsk and ftsl).
have been cloned and the DNA sequenced.
confirmed
genetic
genes(ftsQ,ftsA,ftsZ)
studies
that
are closely
linked,
These genes
DNA sequence analysis has
three
of
that there
these
fts
is no strong
transcription terminator between the genes, and that promoters exist
upstream of each of the genes.
However, information concerning the
regulatory and morphogenetic functions of these genes in the septation
process at the molecular level is only starting to be accumulated.
At
present the biochemical roles of the FtsQ, FtsA, and FtsZ proteins in
septum formation are not known.
1
2
Microscopic studies of cells and physiological analyses of cell
division
mutants
morphogenetic
division
division,
have
suggested
pathway.
mutants,
the
that
Phenotypic
such
as
cell
percentage
the
properties
shape,
of
septation
of
the
persisting
process
is
conditional
amount
of
constrictions
a
cell
residual
at
the
restrictive temperature and the pattern of division recovery after a
shift back to the permissive temperature have suggested that the cell
division genes are activated in the order FtsZ-FtsQ-FtsA-FtsI.
example,
conditional
lethal
nonpermissive
temperature
constrictions
at
mutants
do not
potential
cell
of
divide
ftsA
but
division
when
filament
sites.
shifted
to
with partial
Thus,
protein appears to function after septation initiation.
For
the
FtsA
The ordered
functions of these cell division genes may be controlled at the level
of
new
proteins.
transcription
or
the
activation
of
previously
existing
It is probable that both events occur but the current
evidence strongly favors the protein activation model as the primary
mechanism which commits a cell to septum formation.
In this thesis,
synthesis and
I attempt to verify the hypothesis that the
invagination of surface
layers
constituting
the new
septum require the formation of a structure which has been called the
"septalsome".
The septalsome
is a multienzyme-protein complex
in
which the known septation proteins (FtsZ, FtsQ, FtsA and Ftsl) and
perhaps others would initiate and control the septation steps.
This
septalsome would contain both specific peptidoglycan synthesizing and
modifying enzymes plus envelope
structuring proteins essential
for
3
septation. This organelle would thus ensure the precise coordination
of the enzymatic activities and localization of the septation proteins
within the cell envelope.
In this study, I have focus primarily upon the FtsA protein as a
potential septalsome protein.
Since the amount of FtsA within a cell
is small, the construction of
FtsA fusion proteins were necessary.
The location of these fusion proteins in dividing wild type cells was
accomplished by assaying 0-galactosidase activities of the FtsA-LacZ
fusion proteins
investigate
in soluble
and membrane
location of authentic
fraction of cells.
FtsA protein,
a
To
plasmid which
overproduced the FtsA-LacZ protein was constructed and fusion protein
was purified and injected into rabbits for production of polyclonal
antibodies.
Western blotting experiments were conducted with these
antibodies on cell membrane fractions. Direct visualization of FtsA
protein at septation sites was done by immunoelectron microscopy.
In
addition,
in
various
promoter
fusion
plasmids
were
constructed
attempts to investigate the regulation of the cell division genes,
ftsk and ftsZ.
Literature Review
I.
Identification of cell division genes.
A number of genes essential for cell division in Escherichia
coli have been mapped to the 2 min region of the genetic map(36,3).
These genes have been mainly identified as temperature sensitive(ts)
lethal mutations, so called fts(filamentation temperature sensitive)
phenotype,
that
result
in multi-nucleated
filamentous
growth
and
eventual cell death at the nonpermissive temperaturedl,77,94,120).
The ftsZ(sulB) gene and two neighboring cell division genes, ftsQ and
ftsA in this 2 min region(94,96,109,129,130) are part of a large cell
envelope-cell division gene cluster (Fig. 1).
That is, in addition
to fts genes this 2 min region encodes for murein genes( 8 genes)
which specify enzymes required for the peptidoglycan synthesis of the
cell wall,
cell permeability-cell separation gene, envA(7), and the
secA gene which is essential in protein export across the cytoplasmic
membrane(90).
This close clustering of cell envelope genes and cell
division genes suggests possible coordinated interactions among the
gene and gene products that result in
septum formation. The
peptidoglycan synthesis and
ftsZ, ftsQ, ftsA and ftsl gene in this cluster
encode proteins that probably are required to initiate,
complete a cell septum.
form and
They have been hypothesized to act in a
morphogenic pathway(9,Fig. 2) although little is known about their
specific functions or biochemical activities in septum formation.
FtsZSk
is
a
temperature
sensitive(ts)
mutant
that
at
the
nonpermissive temperature forms multinucleated nonseptated filaments
lacking any persisting or newly initiated septal constriction(77).
When
double
mutants
that
were
4
both
temperature
sensitive
for
5
elongation(rodA)
and
septation
were
analyzed(9),
the
results
indicated that the formation of any septum-like constriction required
expression of the FtsZ gene product. That is, FtsZ acts earlier than
FtsQ,
FtsA,
or
FtsI(PBP3).
synthesis(127)
revealed that
was
be
found
to
(septum sites)
nonconstricting
Analyses
ftsl
ftsZ84
new
peptidoglycan
radioactive DAP(Diaminopimellic
incorporated
of
of
near
newly
initiated
acid)
constriction
mutant
filaments,
but
was
not
found
mutant
filaments.
In
addition,a
in
recent
morphogenic and physiological study(113), investigating the formation
of
septal
constrictions
and
the
amount
of
cell
division
of
ftsZ,ftsQ,ftsA, and ftsl mutants after a temperature shift from 28°C
to 42°C, has suggested that the functioning
ofthese cell
division
genes is in the order FtsZ- FtsQ- FtsA,Ftsl.
Genetic
demonstrated
and
biochemical
that
the
FtsZ(SulB)
evidences
gene
(45,46,56,57,58)
product
SOS(DNA damaged) induced cell filamentation.
is
the
have
target
of
SulA(SfiA) synthesis is
induced following DNA damage as are DNA repair enzymes that are
regulated by the LexA-RecA regulatory circuit(86,71).
SulA protein
is labile with a half-life of 3 min in wild type cells(64).
In a Ion
mutant(101,180) SulA is more stable with a half-life near 20 minutes,
which enhances its inhibitory effects and leads to filamentation and
death
of
the
Ion
irradiation(56,58).
mutant
after
SOS
induction
such
as
UV
SulB(sfib) mutations (like sulA(sfiA) mutations)
were isolated as second site mutations that confer UV resistance to a
Ion mutant. The suIB locus was found to be identical to ftsZ gene
locus(45,73,64).
The hypothesis that FtsZ
protein is supported by the findings that
isthe target of
SulA
the overproduction of FtsZ
6
can suppress the SOS-induced lethal filamentation in Ion mutants(75),
and that biochemical studies suggest direct interactions between the
FtsZ protein and SulA proteins because the half-life of radioactively
labelled SulA in maxicells was increased when cells contained the
wild type ftsl gene but not sulB mutation(65).
The inhibition of
cell septation by SulA interacting with FtsZ is reversible in wild
type cell since SOS-induced filaments eventually divide even in the
absence of de novo protein synthesis(80).
It has been hypothesized that a possible role of FtsZ protein
is as an element of the cell division apparatus to which the cell
transmits information about growth conditions(67). This role for FtsZ
is based on studies with a ftsZ(sfiB114) mutant.
unable to interact with SulA(64) but it
This mutant
is
had a longer cell septation
delay( 50 to 60 min.) compared with wild type (20 min) after a shiftup
from glucose minimal
medium to
complex media,
and
it had
a
shortened cell septation delay (10 to 16 min) after a nutritional
pulse (a shift up to complex media followed rapidly by a return to
glucose minimal medium).
When the level of ftsZ gene expression was increased, minicell
formation and increased septation that results in
slightly smaller
cells have been observed. Ward and Lutkenhaus(123) found that just a
two to seven-fold increase of FtsZ synthesis induces polar divisions
producing minicells.
These minicells were not formed at the expense
of
as
normal
divisions,
they
are
in
a minh
minicell
producing
mutants, but were the result of increased septation. The small cell
size of cells containing multicopy-plasmids specifying ftsZ
appears
to be because medial septal invaginations are initiated earlier in
7
the cell
division cycle.
However,
specifying multicopy-plasmid
into
the
introduction of a ftsZ
minb mutants
results
in
just
increased polar division producing more minicells with normal medial
divisions(123).
This
observation
suggests
between the FtsZ and minB locus products.
that
FtsZ
protein
is
involved
in
potential(medial) division sites.
the
possible
interactions
These findings suggest
maturation
stage
of
the
The FtsZ-induced production of
minicells was suppressed in cya mutant(27). cya mutant cell phenotype
is
short
rods
because
cAMP-CRP(Catabolie
necessary for longitudinal cell growth.
with
earlier
observations(55)
that
Regulatory
Protein)
is
This finding is consistent
introduction
of
cya
or
crp
mutation into a minb minicell strain suppresses minicell production.
The function of cAMP-CRP in minicell formation is postulated to be
between the division machinery and PBP2(penicillin binding protein
2).
Minicell strains are deficient in PBP2.( see Sect2 for role
PBP2).
The ftsZ(sulb) gene has been subcloned and sequenced (95,129)
and identified as a 45 Kdal protein that is membrane associated(53).
Ward and Lutkenhaus have purified the FtsZ protein and have made
antibodies to it (75).
They found that when cells were screened by
Western blotting a FtsZ analogue was antigenically conserved in all
tested
procaryotes
both
Gram-negative
and
Gram-positive.
Only
closely related enterobacteriaceae, however, hybridized with an E.
coli ftsZ DNA probe by Southern blotting.
of
Furthermore,the
induction
minicells phenotype was observed in other strains transformed
8
with multicopy-plasmids containing ftsZ gene, demonstrating that the
E.
coli
FtsZ
protein
could
function
in
several
other
bacterial
species(24).
FtsQ
oscillation
mutant,
T0E1,
enrichment(11).
Its
was
obtained
temperature
remedial by 1% sodium chloride (as is several
by
temperature-
sensitivity
is salt
other fts mutants,94).
The ftsQ gene has been subcloned and sequenced(130), from which the
DNA sequence
predicts
that
FtsQ
protein
is
31.4
Kdal
in
size.
However, no radioactive polypeptide has been identified as the FtsQ
gene product even though several in vivo labelling procedures were
tried that included maxicell and minicells containing ftsQ encoding
plasmids
phage.
and
UV
irradiated
cells
infected
with
ftsQ transducing
Thus, FtsQ protein is likely to be either synthesized in very
small amount or is extremely labile or both.
Filamentous cells of
ftsZ and ftsQ mutant at the nonpermissive temperature have similar
phenotypes.
Both mutant filaments are resistance to 0-lactam lysis
and both quickly have septa
temperature, 30°C (113).
constrictions
However, filaments of ftsQ mutant can have
indicating
initiation step.
after a shift back to the permissive
that
septation
can
continue
pass
the
Thus the FtsQ gene functions after FtsZ gene but
before FtsA, Ftsl genes.
Directly
blocking
DNA
synthesis(
e.g.
dnaA(ts),
dnaC(ts),
dnaB(ts) etc.) causes inhibition of septation and produces transient
filamentous cells(32).
pathway:
This SOS independent division inhibition(TER
18,37,61,62,115,117)
can
result
in
short
uninucleoid
filaments that can produce normal size cells from their end which are
DNA-less.
This anucleated cell production
requires a functional
9
cAMP-CRP complex and is affected by suIB mutations which suggests an
involvement of FtsZ in
the TER pathway.
of
involves
the
cAMP-CRP
division) by
CRP,
since
may
The division promoting role
activation
of
FtsZ(that
promotes
protein(s) or other metabolic factors(67) by the cAMPFtsZ
expression
is not
directly
regulated
independent
division
by
cAMP-
CAP(27).
The
reversal
of
the
SOS
inhibition
requires a short period of protein synthesis at the end of a round of
chromosome replication(66).
termination
division
protein
and
its
FtsA has been proposed as a possible
because
it
synthesis
controls
appears
to
late
be
events(35,118)
coordinated
with
of
DNA
replication in the TER pathway(27,115,117).
The gene product of FtsA has been postulated to be a membrane
protein(53) and
a structural component of the septum(116).
The
ftsA gene has been cloned and DNA sequenced(95); and identified as a
50 Kdal polypeptide on SDS-PAGE of extracts of in vivo labelled cells
containing
ftsk on
plasmid
or
a
X
transducing
phage(76).
The
expression of FtsA is very low, even when amplified by introduction
of a multicopy plasmids encoding ftsA(109,129).
Possible protein
interaction between FtsA and PBP3(119) suggests an involvement of
FtsA in peptidoglycan synthesis specific for septum formation. The
properties
combination
of
double
with
mutants
either
the
harboring
ftsQl,
the
ftsAlO,
Ion
ftsA\2,
mutation
or
in
ftsZSh
mutations suggests that FtsQ, FtsA, and FtsZ proteins also posibly
interact with each other and perhaps form a molecular complex(29).
FtsKpbpb or sep),which is closely linked but separate from
ftsQAZ locus, has been cloned and sequenced(87).
Its gene product
10
(60 Kdal)
is present
membrane(103).
Ftsl
protein,PBP3(see
codes
Sect.2),
transglycosylase
50
for
which
activities
vitro(14,31,60).
inhibition of
in about
a
per
minor
posesses
on
Mutations
copies
cell
penicillin
both
cell
binding
transpeptidase
peptidoglycan
affecting
in the
substrates
Ftsl(94)
or
and
in
selective
PBP3 by 3-lactam antibiotics such as cephalexin(58)
impair cell septation and produces filaments.
PBP3 is the only known
protein target for 3-lactam antibiotics that involves cell septation.
3-lactam induced cellular lysis at potential division sites occurs
only after the action of FtsZ,Q,A division genes. The sensitivity of
fts
mutants
to
3-lactam
lysis
plus
other
morphogenic
and
physiological studies (113) are also consistent with ordering Ftsl
function
after
evidence
from
FtsZ,FtsQ,FtsA
function.
autoradiographic
Furthermore,
analysis
of
indirect
peptidoglycan
synthesis(127) suggests the PBP3 is not involved in the initiation of
constriction but rather in its proper completion.
Recently, the envA gene located immediately downstream of ftsZ
has been sequenced and
mutagenesis(7).
found to be
an essential gene by insertion
The phenotype of a single non-temperature sensitive
envA mutant(envAl)
is
filamentous
cell
chains that
have
inner
membrane separation but not outer membrane-cell wall constriction.
The envA defect is probably due
to decreased N-acetyl muramyl-L-
alanine amidase activity(128).
Minb at 26 min on the genetic map seems to inactivate old
septal
sites
(28,114),
since
minb
mutants
produces
minicells.
Rothfield's group(13) has recently cloned the minb locus and
that
minb locus codes
found
for several gene products indicating it is
11
probably a multi-gene operon.
the
They have suggested that the role of
minb locus is localization of the septation sites, since
under­
expression of minB locus results in minicell production.
Another fts gene cluster maps at 76 min and contains ftsY,
ftsE, and ftsX(48).
from
ftsY.
The
Gene
ftsS is closely linked
location
of
the
heat
shock
to, but separate
regulatory
rpoHihtpR/hin), immediately downstreamof this ftsYEX cell
gene,
division
operon, suggests a possible function of the ftsE cluster is linking
the cell division process
to some of
the majorglobal regulatory
networks of the cell( e.g. the SOS or
DNA repair regulon, the heat
shock regulon,
the stringent
level during division(22,88)
bacterial
ATP-binding
response regulon).
Changes of Ca++
and homology of the FtsE protein to
proteins
involved
in membrane
transport(50)
also suggests a role of FtsE in cell cycle-dependent ion transport.
Other fts mutants that may block division includes ftsB(48 min)
and ftsH(69 min).
Ftsb mutant has been shown to be allelic with
nrdB(68,lll) which is involved in the synthesis of DNA precursors.
Cell division inhibition of this mutant
is growth rate dependent
suggesting indirect involvement of FtsB in the septation process.
.FtsH was previously identified as a gene involved in some initial
stage of septum formation due to a slow stop of cell division at
42°C(1).
A
possible
role
of
FtsH
protein
is
to
regulate
the
synthesis or stability or/both of PBP3(41). This was suggested by a
recent finding that there was absence of PBP3 in cell envelopes of an
ftsH
mutant
at
42°C
even
when
overproducing PBP3(ftsI) plasmid.
the
mutant
strain
contained
an
The function of ftsM, which maps
near leu, appear to be in SOS-associated repair.
The ftsM mutant are
12
affected in Weigle reactivation and its expression appears to be
regulation by LexA (38,39).
A possible interaction of FtsM with FtsZ
protein has been suggested because there was amplification of the
ftsZ7\ mutation phenotype when in the ftsMl mutation was present in
the mutant strain(12,89).
II.
Roles of penicillin binding proteins in morphogenetic cycle.
Penicillin binding proteins (PBPs), located in the intermembane
structures(4,97)
represents
a
within
set
of
the
cell
proteins
envelope
of
Escherichia
responsible
for
coli,
peptidoglycan
biosynthesis involved in cell shape, elongation and septation(55).
These proteins are the actual enzymes that catalyze the insertion of
new material into the peptidoglycan sacculus and are engaged in the
control of cell elongation(106) and cell division(14,63).
PBP1A and IB are transpeptidases present at 200 molecules per
cell(104)
which
are
responsible
peptidoglycan(PG) net.
for
the
crosslinking
of
the
Inhibition of the function of PBP1A and IB by
mutations or cephaloridine(
a 8-lactam antibiotic)
results in an
inhibition of PG synthesis causing lethal cell lysis.
PBP2(pbpA
gene
product)
is
involved
in
the
control
of
elongation of the rod-shaped sacculus and indirectly cell septation.
Mecillinam,
a
8-lactam
antibiotic
that
specifically
binds
PBP2,
inhibits cell elongation and produces spherical cell (106) and that
cannot
divide(63).
It has been proposed
that
the
lethality of
mecillinam bound to PBP2 is primarily the result of this septation
inhibition
(26).
Most
mecillinam-resistant
mutant
cells
were
13
mutations In pbpA. and rodk genes.
These mutants have a spherical
cell phenotype in the absence of mecillinam and they apparently grow
by constant septation(27).
in cya, crp genes.
Other mecillinam resistant mutants were
These mutants have a rod shaped cell phenotyp in
the absence of mecillinam.
The cya and crp mutants formed filaments
when DNA synthesis was interrupted, since the functional cAMP-CRP
complex is required for the production of anucleated cell (see sec
1).
Recent cya expression studies using a cya-lac transcriptional
fusion plasmids(120) have revealed that cya(adenyl cyclase gene) is
transcribed during cell elongation and its transcription is repressed
during cell septation.
Interestingly, it was found that increased
expression of cya inhibits cell division resulting in filamentous
cell growth.
These data support a possible role of the cAMP-CRP
complex in cell elongation and suggest cAMP-CRP may have a negative
function on cell septation.
The fact that cya and crp mutants
are
mecillinam resistant suggest that the cAMP-CRP regulates some aspects
of
the
interactions
between
PBP2
and
the
septation
apparatus.
Furthermore, the inhibition of lateral wall elongation by mecillinam
stimulates
suggests
septation
that
crp(ts),ftsk
cAMP-CRP
mutant(19).
complex may
be
This
regulating
observation
the
transition
between peptidoglycan synthesis of lateral wall elongation and septum
formation.
PBP3( the gene product of ftsl, pbpb, sep) is a complex protein
of
60
Kdal
activities
with
which
transglycosylase
is
formation(14,31,60).
involved
Septal
in
and
transpeptidase
murein
peptidoglycan
synthesis
synthesis
enzymatic
and
is
septum
inhibited
during the period of actual septation by selective inhibition of PBP3
14
or FtsI mutations.
Biochemical data(31) has revealed that PBP3 is
responsible for viability when there is a under limitation of
pentapeptides,
nonseptal
since
PBP3
can
transpeptidations.
use
the
Other
available
biochemical
murein
tripeptide
studies(31)
for
have
demonstrated that at least part of the septal peptidoglycan, which
contains increased amounts of cross linked peptidoglycan with a high
ratio of tripeptide cross linked subunits,
is synthesized by PBP3
using UDP-N-acetylmuramyl tripeptide-substrates as acceptors.
PBP4
a
49
carboxypeptidase
Kdal polypeptide
and
DD-endopeptidase
transpeptidase activity in vivo.
secondary
that has
transpeptidation
been
shown
activity
in
to
vitro
have
and
PBP4 is believed to be involved in
resulting
in
additional
peptidoglycan
cross linking, that in a way converts new murein into old murein(55).
PBP5(dacA)
is
a
42
Kdal
polypeptide
that
has
DD-
carboxypeptidase activity which is high just before septation(8).
PBP5
is
subunits
responsible
into
substrate
tri-
for
for
and
PBP3
converting
peptidoglycan
tetra-peptide
subunits,
and
septal
pentapeptide
the
peptidoglycan
preferred
synthesis.
Overproduction of PBP5 results in spherical cells, which is the same
phenotype exibited by PBP2 mutants.
A recent model(8,82,85) for the
morphogenetic cycle in E. coli invokes a shifting balance in the
relative activities of PBP2 and PBP3
that is driven by periodic
changes in the activities of D-alanine carboxypeptidase(PBP5). This
model
does
explain
overproduced
or
pentapeptide
chains
required
for
the
PBP2 is
cell
into
spherical
cell
mutated.
tetrapeptides
elongation
because
phenotype
Increased
when
conversion
suppresses
PBP2
PBP5
is
of
crosslinking
requires
nascent
15
peptidoglycan chains with complete pentapeptides.
The location of
dacA(PBPS) gene's chromosomal location is near gene locus with rodk,
pbpA genes at 15 min on the E. coli genetic map (105,108).
The
low
numbers
of
PBP3(50
molecules/cell)
and
PBP2(20
molecules/cell) molecules which are produced at a constant rate in
each cell
suggest
cycle
that
from
the
a small
balanced
number
of
activities
mRNA
molecules(104,126)
between
PBP2
and
PBP3
is
obtained by the modulation of their enzymatic activities rather than
by
their
f£sZ84(ts)
synthesis.
mutants
Biochemical
exhibit
a
studies(84)
low
have
activity
shown
of
that
D-alanine
carboxypeptidase at 42°C when the cells are filamentous, suggesting
FtsZ may be a modulator.
In addition, other data(31,42) point to
FtsA and FtsH as possible modulators of PBP3 either regulating the
enzymatic
activity
and/or
protein
stability.
A
possibility
for
protein-protein interaction between PBP3 and FtsA was suggested by
the increase resistance of ftsA mutants to the lytic action of Plactam
antibiotics
and
[125I]benzypenicillin
by
to
nonpermissive temperature.
the
PBP3
finding
in
ftsA
of
decreased
mutant
binding
strains
at
based on decreased binding of
PBP3 to 125I-ampicillin in a strain carrying the sulB mutation.
PBP3 and RodA(lO)
combination of
activities
mutation in both rodA or
of
the
cell
In
proteins may also interact because a
mutants with normal shape and division.
the
the
Possible interactions of PBP3 with FtsZ
have also been implied(31), which was
addition,
of
division
pbpB gene can results in
These findings indicate that
genes
and
the
enzymatic
activities of PBPs are highly coordinated and regulated, possibly as
part of a multimeric bifunctional complexes for both cell elongation
16
and septum formation.
be,
in
turn,
These multimeric bifunctional complexes could
modulated
by
multiple
factors
linked
to
global
regulatory metabolic circuits at discrete places(possibly potential
division sites) within the cell envelope of E. coli.
II.
Periseptal anulii and potential division sites.
Donachie et al.(36) observed that in dnaA(ts) mutant at the
nonpermissive temperature(42°C)
unreplicated nucleoid at potential
division
block
sites
production of
resulted
in
a
of
cell
septation
short anucleoid filamentous cells. Normal
less cell were
produced from these
anucleoid
and
the
sized DNA-
filaments when the
transient chromosomal replication blockage was removed by returning
these cells to the permissive temperature.
that
the
cell
continues
This finding indicated
to develope potential
division sites of
unicellular length in the absence of new chromosomal replication(50).
Thus
DNA
finding
occurs
replication
and
cell
septation
can
be
uncoupled.
This
also indicated that the initiation of the septation process
before
completion
of chromosome
replication.
Jones
and
Donachie(66) and by Holland(52) have proposed that activation of cell
septal
division
replication.
sites
occur
after
the
termination of
chromosome
In fact, Donachie and Beggs(34) found that potential
division sites couldbe detected by their sensitivity to
penicillin
and that these sitesappear to be present at regular intervals early
in the cell cycle.
Teather et al (114) proposed that the synthesis
of quantumly produced division factor
frequency of the cell septa.
controled the location and
This hypothesis was to explain why a
17
normal potential division site was lost in the miriB mutant, in which
the synthesis of a minicell results in the parent cell usually being
two cell lengths.
Recently, Rothfield's group observed these potential division
sites by TEM in
as
serial sections of plasmolized bacterial cells(78)
concentric rings of tightly bound inner-outer membrane [commonly
called
adhesion
zones(5)].
"periseptal annuli".
They
called
this
organelle
the
The periseptal annuli flank the constriction
site around the potential septa and they appear to act as an innerouter
membrane
between
gasket
compartmentalizing
developed
a
membrane
isolation
of a membrane fraction, called outer membrane lightCOMjJ,
Biochemical
involved
fractionation
this
studies(59)
in
the
Ishidate
procedure
inner-murein-outer
demonstrated
translocation
that
et
space
dividing
contains
3).
periplasmic
the
which
cells(Fig
the
al.(59)
that
enables
membrane
this
of
have
0ML
newly
the
fraction.
fraction
is
synthesized
lipopolysaccharides and peptidoglycan synthesis. Additional evidence,
supporting the model
that
the
periseptal
annuli is an cellular
organelle associated with cell septation, is that the 0ML fraction
was increased in cell division mutants, IkyD and cha. Both mutants
are
defective
formation(21).
in
outer
Incomplete,
membrane
nascent
invagination
during
septum
annuli
do
extend
that
not
completely around the cell were also observed at nonseptal regions
along growing celIs(25).
These nascent annuli appear to be generated
from complete annuli already in position at the midpoint of newborn
cells,
and
were
displaced
laterally
during
cell
elongation
to
position at 1/4 and 3/4 cell lengths that become the midpoint annuli
18
of newborn cells of forthcoming divisions(25).
These newly generated
annuli have been hypothesized to be the cell structure involved with
chromosome partitioning during the cell division.
The finding of periseptal annuli at potential division sites in
a divC mutant filament(26), in which cell division was blocked before
any septal invagination, indicated that divC gene product acts after
biogenesis, maturation and
localization of the periseptal annuli.
Thus,the formation of the periseptal annuli precedes the initiation
of septal
invagination.
The
identification
of
a second
division
related structure, septal attachment sites, SAS(79), suggest that it
may be a first step in septal invagination.
The miriB locus has been proposed(13) to be involved in the
localization of the division septum.
The single polar periseptal
annulus, which remains at the cell pole in each daughter from the
preceding division event, could be the foci for the abnormal polar
division and minicell formation in miriB mutant strains(13,29).
IV.
Preliminary studies on the regulation of ftsQAZ gene expression.
The 2 min region of E, coli genetic map including ftsQAZ genes
contains a large cluster of 14 genes, approximately 20 kilobases,
involved in cell envelope growth and division (96,Fig.l).
The close
clustering of these genes suggest a possible coordinate expression
and/or interaction between the genes. The cloning of DNA from this
region and the
sequencing and subcloning of DNA fragments into
promoter assaying have enabled the construction of a transcription
map of the region(95,96,109,129,130).
Although the contiguous genes
19
between dd!(D-ala:D-ala ligase) and envA are transcribed in the same
direction,
this regions forms an atypical operon with overlapping
transcriptional
units
since
each
gene,
when
expressed from its own promoter(110,130).
subcloned,
can
be
The promoters (Pz3 and
P«) of the ftsZ gene are within the coding sequence of the upstream
ftsA gene(110), the promoter of ftsk in turn is within the coding
sequence of upstream ftsQ gene(95), and the promoter for ftsQ may be
within
its
upstream
gene
neighbor.
SI
mapping
(Corton
and
Lutkenhaus, unpublished) and the used of a terminator-probe vector
has
recently
independent
revealed
the
location
of
a
typical
strong
rho-
transcriptional terminator just downstream of envA(7).
The absence of strong transcriptional terminators between ddl and
envA suggests that transcription originating from ddl could continue
through
to
transcripts
envA.
are
However,it
produced
different metabolic signal
form
remains
this
gene
to
be
cluster
determined
in
what
response
to
in vivo.
A possibility is that gene
expression involves promoter specific
transcription initiation and
RNA polymerase transcriptional pausing.
A computer search of the
published DNA sequence of the ftsQ-ftsA region(95) has identified two
potential NusA sites with a possible DNA sequence that could form a
stem-loop structured!. Gayda, unpublished data), one at the beginning
of the ftsQ gene sequence and the other near the ftsZ promoter Pz3.
NusA specific DNA sequence has been demonstrated to be necessary for
lambda N gene antitermination(43).
The expression study of ftsZ gene has revealed substantial
reduction of ftsZ synthesis when a TnlOOO transposon was inserted
within the ftsA gene(64). Similarly, an
ftsZ encoding DNA fragment,
20
containing only the
two promoter (Pz2 and PZi) cloned into X vector,
was insufficiently expressed to complement an ftsZ mutant. However, a
X ftsZ transducing phage was able to complement a ftsZ mutant(74)
when it contained additional upstream DNA including the promoter(Pz3)
within the ftsA gene but not the entire
Sullivan and Donachie(109) found
and Pz3
ftsA gene coding region.
that the transcription from Pz2Pzi
when togetherwas more than
the sum of their separate
promoter activities.
Similarly,there is evidence(33) that upstream
DNA
the ftsA
in
addition
to
promoter is
required
for
proper
expression of the ftsA gene. These data suggest that regulation of
the ftsQAZ genes is not simple but complex.
Transcriptional expression of ftsQAZ encoding region also seem
to
involve
promoters.
different
regulatory
interactions
amoung
the
various
The amount of FtsZ per cell was invariable over a 10 fold
range of cell mass, thus suggesting that ftsZ expression is subject
to some type of autoregulation(36,65).
found
to
expression
be
increased in a
may
be
negatively
Transcription from Pz3 was
cya mutant,
regulated
which
by
suggested
cAMP-CRP
ftsZ
complex.
However,(as previously mentioned) a recent study(27) has demonstrated
that cAMP-CRP does not regulate ftsZ gene expression but that the
increased
small cell
FtsZ protein inthe cya mutant
size of
the
cya mutant.
was a compensation for the
Transcription from Pz3 was
increased as much as 10 fold when ftsA had a nonsense mutation and to
a lesser extent in mutants of ftsl, ftsQ and ftsZ,
Post-transcriptional processing and/or modification of the Fts
gene products have not been reported.
Materials and Methods
I. Bacterial strains, plasmids and phages.
The E. coli K-12 strains, plasmids and phages used in this study
are listed in Table 1 and 2.
100 yl of A1098(10 X 10® plaques forming units) that contains a
mini-TnlO transposon were mixed with 100 yl of an overnight culture of
strain RGC103-9(sulB9,Ion).
After 20 min at room temperature the
mixture was plated out on a YET-plate containing tetracycline(12.5
yg/ml).
YC99
was
Tetracycline-resistant (TetR ) colonies were pooled.
constructed
by
Pl-mediate
transduction
of
Strain
RGC123
to
tetracycline resistance and nitrofurantoin (NF) resistance (to select
for su2B9 allele)
from such a random TnlO bacterial pool.
SulB9
allele was found to be 90% cotransduced with TnlO in YC99.
YC100 was
constructed by PI mediated transduction of ftsAlO allele
linked to
TnlO
(ftsA:Tnl0)
into strain AMC290
by selecting
resistance and temperature sensitivity.
for tetracycline
Strain YC200 was obtained by
Pl-mediated transduction of Lac+ allele from Hfr3000 into AMC290 by
isolating blue colonies on minimal lactose plates containing X-gal(40
yg/ml) after 3 days incubation at 30°C.
II. Media and growth conditions.
E. coli K-12 strains were routinely grown on complex medium, YET
(per liter): lOg Bacto-tryptone(Difco), 5g Bacto-yeast extract(Difco)
and lOg NaCl.
The final pH was 7.0.
YET agar plates included 15 g/1
Bacto-agar(Difco). Low salt complex medium, TEY was identical to YET
21
22
but without any NaCl. Other media used for E. coli growth included:
(i)
M9 minimal
medium(83) which consisted of minimal
salts
(per
liter): 6g Na2HPOA, 3g KH2P0A, 0.15g NaCl, and lg NHACl,to which after
autoclaving
and
cooling were
carbon source was 0.2% glucose
added
plus
lOg glucose
uridine per liter.
and
and O.lmM CaCl2,the
and amino acids and vitamins added as
required. The final pH was 7.4.
salts
2mM MgSO*
(ii) KU-medium(lOO) was M9 minimal
lOg NZamine(Sheffield
products),
0.5g
(iii) Sulfate free Hershey medium(lOO) that was
(per liter) 4g glucose, 1.0 mg thiamine(B1), 5.4 g NaCl, 3g KC1, l.lg
NH*C1, 15 mg CaCl2-2H20, 203 mg MgCl2-6H20, 0.2 mg FeCl3-6H20, 87mg
KH2POa, 12.lg Trizma base(tris[hydroxymethyl]aminomethane).
The final
pH was 7.0. (iv) Lactose agar plates(83) consisted of (per liter) 10.5
g K2HPOa, 4.5 g KH2POA, 0.5g
Bacto-agar,
(NHA)2S0A, 0.5 g sodium citrate,
to which after autoclaving,
15g
1.0 ml of 20% MgS0A-7H20,
0.2ml of 5% thiamine, 10 ml of 20% lactose, 5ml of 2% amino acids as
required were
added.
Final
pH was
7.0 without adjustment.(v).
L
medium consisted of(per liter) 10 g Bacto-tryptone, 5 g Bacto yeast
extract and 5 g NaCl.
Carbenicillin
kanamycin(kan,25
(carb,25
yg/ml,
yg/ml),
and
tetracycline(tet,12.5
yg/ml)
5-bromo-4-chloro-3-indoly-(5-D-
galactopyranaside(X-gal, 40 yg/ml) were added when needed.
Overnight cultures at 30°C were diluted approximately 100 fold
in fresh media and grown to 0D6Oo = 0.1 to 0.5 before cells were
challenged by experimental conditions such as a temperature shifts.
The permissive temperature for the ftsZ, /tsA, ftsQ, ftsl mutants was
30°C and the restrictive temperature was 42°C.
23
ftsk mutants, YC100, was unable to grow on TEY plates at 30°C,
and exhibited temperature sensitive phenotype on YET media at 42°C.
ftsl mutant,AMC493, was sensitive to growth at 42°C on both YET and
TEY media.
ftsZ mutant,AMC419, and ftsQ mutant,T0E1, was temperature
sensitive only on TEY (no salt) media.
Growth of ftsZ, ftsQ mutants
on YET plates incubated at 42°C results in colonies.
Strain YC200( laC") was induced for expression of 0-galactosidase
with ImM IPTG(isopropyl 0-D-thio-galactopyranoside) at 0D6Oo = 0.12
for 2 hrs.
Ill Enzymes and chemicals.
Restriction endonucleases were obtained from either New England
Biolabs,Inc., or Bethesda Research Laboratories(BRL).
T4 DNA ligase,
bacterial alkaline phosphatase and the large fragment of DNA poll
(Klenow) were obtained from BRL.
p-chloromercurophenyl sulfonic acid(pCMS), used as an inhibitor
of contaminating nuclease activity, DL-dithiothreitol, and Trizma Base
(tris[hydroxymethy] aminomethane) were purchased from Sigma Chemical
Co.
Alkaline-phosphatase
linked
goat
anti-rabbit
imunoglobulin
G
antibody (Western blot AP system, W3930) was obtained from Promega.
The goat anti-rabbit-immunoglobulin G antibody labelled with lOnm gold
particles was purchased from Jenssen (Pharmaceutical).
0-lactamase
substrate,
PADAC([l-(thienyl-2-acetamido)]-3-[2-(4-
N,N-dimethylaminophenylazo)pyridium methyl]-3-cepem-4-carboxylic acid)
was purchased from Calbiochem(Behring Diagnostics).
24
IV.
Methods of DNA preparation
a.
Large scale plasmid DNA isolation.
Plasmid DNA was prepared by the method of Robert Kolter with
some modifications.
Five mis of an overnight culture of cells that
were grown in L broth plus 40 pg/ml ampicillin were used to inoculate
1 liter fresh L+amp. The culture was grown at 37°C (or 30°C) with
shaking(200 rpm) to a optical density of 0D6Oo=0.4.
amplified
continuing
in
the
cells
incubation
by
adding
overnight
with
Plasmid DNA was
chloramphenicol(150mg/l)
shaking.
The
cells
and
were
harvested by centrifugation for 5 minutes at 5,000 X g which yields a
soft pellet of cells.
These cells were then resuspended in 5 ml of
50mM Tris-Cl(pH 8.0) with 25% (w/v) sucrose.
0.5 mis of lysozyme
(lOmg/ml in a solution of 50mM Tris-Cl,pH 8.0, and 25% sucrose) was
added and the mixture was incubated on ice for 5 minutes.
Then, 0.5ml
of 0.25M EDTA (pH 8.0) was added and further incubated on ice for
another 5 minutes.
Next, 5 ml of Triton X-100 lytic mixture (50mM
Tris-Cl(pH 8.0),62mM EDTA and 0.4% Triton X-100) at room temperature
was added quickly, mixed, and left at room temperature for 5 minutes
or until a clear lysate was achieved.
Cellular debris was sedimented
by ultracentifugation at 40k r.p.m. for 45 minutes in a Beckman type
75 Ti
rotor.
centrifuge tube.
The supernatant
fluid was
decanted
into
a plastic
One gram of CsCl and 0.1ml of a solution of 5mg/ml
ethidium bromide was added per ml of supernatant.
The protein debris
was then removed by centrifugation at 10K rpm (Sorvall SS34 rotor) for
5 minutes.
The supernatant was transferred to quick-seal polyallomer
tubes and CsCl density gradients were established by centrifugation to
equilibrium at 49k rpm in a Beckman VTi65 rotor for 20 hrs at 15°C.
25
Covalently closed circular plasmid DNA band was visualized with long­
wave ultraviolet light and extracted from the side of the tube with a
number 21 needle and syringe. To remove the ethidium bromide,
plasmid
DNA
in
CsCl
saturated butanol.
solution
was
extracted
6
times
the
with water-
The solution was then diluted with two volumes of
water; the DNA was precipitated by the addition of ethanol at 6 times
the original volume, and solution kept at -20°C for 2 hours.
The
plasmid DNA was pelleted by centrifugation at 10,000 X g, washed with
80% ethanol, dried in vacuo, and resuspended in T.E.(10 mM Tris-HCl,
ImM EDTA, pH 8.0) buffer.
b.
Small-scale (rapid) plasmid isolation.
Small
quantities
of
plasmid
DNA
were
prepared
for
rapid
screening by the method of Holmes and Quigley(54) with modifications.
5 mis of bacteria that were grown overnight were pelleted at 3000 X g
for 5 min and resuspended in 0.35 ml of STET2 buffer( 8% sucrose,
0.05%
Triton
X-100,
resuspended cells
50mM
25 yl
EDTA,
of
lOmM Tris-HCl(pH
freshly prepared
8.0)).
To
the
lysozyme(lOmg/ml)
was
added, mixed briefly and then the tube was placed in a boiling water
bath for 40 secs. The tubes were chilled in ice bath and immediately
centrifuged at 12,000g for 20 min at 4°C.
The supernatant was drawn,
1/10 vol of 3M sodium acetate (pH 4.8) was added and then an equal
volume of cold isopropanol was added and the solution was placed at 18°C
for
10
min.
The
DNA
was
precipitate
was
pelleted
by
centrifugation in a microcentrifuge for 5 min at 12,000 g and washed 2
times with ether.
The resulting plasmid DNA pellet was resuspended in
50 ul of TE buffer and kept at -18°C.
26
V.
General genetic and recombinant DNA techniques.
PI
transduction
previously
for
described
strain
construction
(80,102).
Similarly,
were
A
performed
as
growth
and
phage
infection were as described (80,102).
DNA
recombinant
techniques
described by Maniatis et al (81).
were
performed
essentially
as
Restriction enzyme digestions were
for 60 min at 37°C in buffers recommended by the manufacturers.
When
DNA was to be digested with more than one enzyme, the enzyme reaction
requiring lower salt concentration was performed first, then reaction
mix was inactivated by heating at 65°C for 10 min, cooled and adjusted
by the addition of salt to meet the requirement of the next enzyme in
the sequence.
10
mM
to
After completion of enzyme digestion, pCMS was added to
inhibit
nuclease
activity
(without
interfering
with
subsequent ligation reaction) before the usual inactivation at 65°C
for 10 min.
Otherwise, the digestion reactions were terminated by
phenol extraction of proteins followed by ethanol precipitation of the
DNA.
In
some
restriction
DNA
enzyme
fragment
sub-clonings,
digestion
was
the
vector
dephosphorylated
DNA
with
after
bacterial
alkaline phosphatase(0.1 unit/yg of DNA) at 65°C for 1 hr. The DNA was
then ethanol precipitated after phenol-chloroform ether extraction,
and then resuspended in TE buffer( lOmM Tris-HCl, ImM EDTA, pH 8.0).
DNA fragments
elution
onto
from agarose
dialysis
membrane
gels
as
were
described
recovered
in
by
section
electro­
VII,
and
purified through either Elutip-D columns according to manufacturer's
specification
or
by
chromatography
on
a DEAE-50
column.
The
DNA
27
fragments were further cleaned by pheno-chloroform-ether extraction
and then ethanol precipitation.
DNA pellet was finally resuspended in
TE(10:1) buffer.
Ligation of DNA fragments were performed in ligation buffer (
66mM Tris-Cl, pH 8.0,
ImM EDTA, 5mM MgCl2, 5mM dithiothreitol, 0.1M
ATP). Optimal ratio of vector DNA to insert DNA was determined as
described by Goodman et al (40).
Plasmid
DNA
described(70).
transformations
were
performed
as
previously
Unless otherwise noted, strain AMC290 was used as the
recipient in the DNA transformations.
The orientation and in some cases whether or not a vector has an
insert DNA fragment was
determined by
restriction enzyme cleavage
mapping of isolated plasmid DNA as previously described(107).
VI.
Bacterial transformations.
Transformation
Lederberg
and
with
Cohen(70)
plasmid
as
DNA
followed
previously
a
modification
described.(101).
cells of appropriate host were prepared as follows.
of
Competent
A 50 ml L broth
culture was inoculated with 0.5 ml of an overnight culture and grown
to
0 D 6Oo
= 0.2.
resuspended
in
The cells were chilled on ice,
50
mis
of
cold
0.2M
MgCl2.
centrifuged and
After
a
second
centrifugation at 4°C, the cell pellet was resuspended in 25 ml of
0.1M CaCl2 and chilled on ice for 20 minutes.
in
the
cold
was
carried
out,
and
the
cell
resuspended in 0.7 mis of cold 0.1 M CaCl2.
mixing
0.1ml
DNA( approximately
,05yg
up
A third centrifugation
to
pellet
was
finally
Transformation involved
lyg)
with
0.2
competent cells and incubating mixture on ice for 30 minutes.
ml
of
To aid
28
DNA uptake the mixture was heat pulsed 30 secs before 30 minute at
37°C
ice
incubation
transformation
and
mixture
after
was
for
diluted
2.5
2
to
minutes
at
10
with
fold
42°C.
L
The
broth
containing 10% glycerol and incubated at either 37°C for lhr or 30°C
for 2 hrs before spreading onto selective agar plates.
were
verified
by
antibiotic
resistance
and/or
Transformants
phenotype( i.e
temperature resistance) .
After intial transformation with a newly constructed plasmid,
the plasmid DNA was isolated and purified as described in section IV.b
and then used in a second transformation to determine the phenotype of
the new plasmid in the appropriate strains.
VII Electrophoretic techniques.
Horizontal
buffer
agarose gels were prepared in TBE electrophoresis
(89mM Tris-borate,2 mM
EDTA,
pH 8.0).
Loading
buffer(6X)
contained 0.25% bromphenol blue, 0.25% xylene cyanol and 15% Ficoll in
dH20.
After electrophoresis,
the agarose gels were stained with a
solution of ethidium bromide(0.5 mg/ml) for 1 hr.
on a U.V. trans-illuminator.
DNA was visualized
Hindlll digested lambda DNA was used as
DNA fragment molecular weight markers.
Sodium
dodecylsulfate(SDS)
polyacrylamide
gel
electrophoresis
(PAGE) was performed utilizing the discontinuous system as described
by Laemmli(69), that was
modified as described previously(44).
The
main gel was polymerized in a solution containing 0.37M Tris-HCl(pH
7.8),
0.1%
SDS,
0.03%(vol/vol)
TEMED,
0.33%(wt/vol)
ammonium
persulfate, with concentration of acrylamide ranging from 10 to 30%
maintaining a N,N'-methylene bisacrylamide-acrylamide ration of 1:173.
29
10% to
30%
linear gradient
acrylamide
gels were
prepared
with
a
gradient maker (Hoefer Scientific Instruments).
The stacker consisted
of 0.14M Tris-HCl(pH
0.15% N,N'-methylene
6.8),
5.7
% acrylamide,
bisacrylamide, 0;1% SDS, 0 .05%(vol/vol) TEMED, 0.05%(wt/vol) ammonium
persulfate.
The electrode buffer at pH 8.5 contained 0.05 M Tris-HCl,
0.384M glycine and 0.01% SDS.
The gels were run at 20 milliamps at
constant current until the dye front reached the gel bottom. Protein
extracts were solubilized by heating at 100°C for 5 min (at 65°C for
30 min for certain membrane fractions) in the presence of 1% SDS,
0.625 Tris-HCl(pH
7.0),
5% 8-mercaptoethanol, 4M urea,
bromophenol blue.
Protein in SDS-PAGE
and 0.015%
were stained and fixed with a
solution of 0.25% Coomassie Brilliant Blue, 43% methanol and 7% acetic
acid and destained in 43% methanol and 7% acetic acid.
VIII.
Extraction of DNA fragments from agarose gels.
DNA fragments were
extracted from agarose gels by
just ahead of the desired band, placing a
cutting
aslot
piece of dialysis membrane
to line the distal side of the slot, filling the slot with IX TBE and
then electro-eluting the DNA into the slot and against the dialysis
membrane.
The DNA was
pulled off the membrane by reversing the
current for 30 seconds, and the buffer in the slot was collected.
The
slot was rinsed two times with buffer which was also collected.
The
DNA
fragments
was
cleaned
by
passage
through
an
Elutip-D
column(Schleicher and Schuell) or by DEAE-52 chromatography.
DNA from
column was concentrated by the addition of one-tenth volume 3M sodium
acetate(pH 4.8) and ethanol precipitation.
30
IX.
Maxicell labelling of plasmid specified proteins.
The procedure used was a modification of Sancar et al. (99,100).
CSR603 was transformed with specific plasmid DNA to be analyzed for
protein
expression.
Three
mis
KU medium
inoculum
of
a plasmid
containing maxicell strain was grown at 37°C to an 0D&oo of 0.2 (2 X
10s cells/ml).
These cells were then diluted 1:50 into
fresh KU medium and grown again to an 0D6Oo of 0.2.
then transferred to a 6 cm petri dish (Falcon).
10 ml of
The cells were
The dish containing
the cells and a small magnetic stirring bar was placed onto a magnetic
stir plate.
A portable 254 nm ultraviolet light(ultraviolet products,
Inc.) was clamped to a stand at a distance of 20 cm from the dish.
The top of the petri dish was removed and the vigorously stirred cells
were irradiated for 30 seconds.
The cells were then transferred to a
125 ml flask, diluted 1:2 with K medium and incubated for 2 hours at
37°C.
It should be noted that all incubation were carried out in the
dark as the bacteria are photo-reactive.
Cycloserine(200 yg/ml) was
added to each cell suspension and the incubation with shaking was
continued for
13 to 14
hours.An addition 200 yg/ml cycloserinewas
added during the final hour incubation.
centrifugation
at 6000 rpm(SS34
The cells were sedimented by
rotor) at 4°C.
The supernatant was
discarded and the cells were washed twice with sulfate-free Hershey
medium(50 ml flask) and incubated with shaking for 1 hour at 37°C. The
plasmid specific protein were labeled for lhr at 37°C
with [35S]-
methionine(New England Nuclear) which was added at a concentration of
5 yci/ml.
6000
The labelled maxicells were pelleted by centrifugation at
rpm and washed
radioactivity.
All
twice with 0.1M NaCl
supernatants
and
to
washes
remove
were
non-specific
treated
as
31
radioactive waste.
The cell pellets were then resuspended in 200 yl
of sample buffer and boiled for 5 minutes to denature the proteins.
The
cpm/yl of
samples
Scintillation counter.
loaded
onto
10%
were
determined
in
a
Beckman
LS6800
Approximately 500,000 to 750,000 cpm/lane was
to
30%
gradient
SDS-Polyacrylamide
gel
and
electrophoresed at 20 mA constant current until the dye front ran off
the end of the gel(approximately 16 hours).
The gel was then dried at
80°C on a BioRad gel dryer. Dried gels were placed directly against
Cronex X-ray film(Dupont).
Development and visualization of the resulting autoradiograms
were as previously described (44,101).
X.
Plasmid constructions.
a.
Ia vivo mini-Mu transposon mutagenesis for the construction
of FtsA-LacZ fusions.
The
procedure
developed
transposon mutagenesis
mutation
of
of
plasmid
MudII1734(Fig.
by
plasmids
pRGC31.
strain
Casadaban(20)
was
used
The
to
mini-Mu
P0II1734(20)
which
for
isolate
mini-Mu
insertion
transposon
1) is a 9.7 Kb derivation of Mudll
confers kanamycin resistance(kanr).
into
M.
phage(20)
used,
that
Plasmid pRGC31 was transformed
contained
a
Mucts
and
mini-Mu,
MudII1734. The strain was heat induced and the resulting phage lysate
was used to infect strain YC99.
Phage transductants that conferred
both the original plasmid resistance(ampicillin from pRGC31) and miniMuresistance to
Phage
kanamycin contained plasmids
transductants
that
expressed
with mini-Mu inserts.
P-galactosidase
activity,
indicating a gene fusion with LacZ, were identified with X-gal on
32
selective
plates(YET
orientation of
with
kan
the mini-Mu
and
amp,
101).
The
location
inserts were determined by
and
restriction
enzyme mapping of the isolated plasmid DNA( Fig 6).
b.
Construction of FtsA-LacZ overproducing plasmid, pYC3.
Plasmid pGW7(30)
is derived
from
the
E.
coli
plasmid
pBR322 and contains a 4.0 Kb EcoRl-BamHl fragment derived from the
bacteriophage lambda control region (Fig. 4).
genes
to be placed under
promoter.
The
PL
the control
promoter
is
of
regulated
This enable foreign
the highly active
by
the
repressor protein (CIasr) contained on the plasmid.
A PL
thermolabile
Cl
Also present is
the A N gene whose product allows for efficient readthrough of rhodependent and rho-independent terminator site.
The
plasmid
pGW7
and
described in section IV. a.
pYC16
were
isolated
and
prepared
as
The plasmid vector pGW7 was completely
digested with BamHi and followed by treatment with bacterial alkaline
phosphatase.
The plasmid pYC16 was partially digested with BartSW
(lu/4yg, 3 min at 37°C).
The 8.5 Kb BamHl DNA fragment containing the
FtsA-LacZ hybrid gene was recovered from an agarose gene and purified
as described in section VII.
The two DNA fragments were
together and then transformed into strain AMC290( Lac).
ligated
Transformants
that grew as blue colonies on X-gal,amp, kan plates were selected.
The
insertion
and
orientation
of
the
8.5
Kb
DNA
fragment
was
determined by restriction enzyme mapping of one isolated plasmid DNA,
which was designated pYC3.
33
c.
Construction of promoter fusion plasmids.
The
ftsZ
regulatory
region
the
appropriate
purified
subcloning
promoter probe plasmid,
gene
fusions
were
restriction
performed
fragment
into
by
the
pKK232-8(16,Fig. 5) that give transcription
fusions to the chloramphenicol acetylase gene (CAT).
pRGC31 was used
as the source of ftsZ DNA.
pYC500 consists of a agarose gel purified 1.7Kb BanMl-Hindlll
restriction fragment from pRGC31 that was ligated into BamHl, Hindlll
digested pKK232-8.
Carta*-, Cmjr(25ug/ml) transformants were verified by
restriction enzyme analysis of BamHl - Hindlll digestions and Bglll
digestions.
In pYC530 the BamHl-Bglll DNA fragment of pYC500 was deleted by
a double
digestion and
ligation.
Again,
restriction analysis
of
carb*, cm* transformants verified the construction.
pYC580 consists of an agarose gel purified 0.5Kb Hindlll-EcoRl
restriction fragment from pRGC31 in which the overlapping DNA ends
were filled in with DNA polymerase I and then blunt end ligated into
Smal ends of pKK232-8 that were dephosphorylated.
One carb*,
cm*
plasmid transformant contained the proper DNA fragment in the desired
orientation based on Hindll and BssHII double digestions.
pYC600 consists of an agarose gel purified 2.2 Kb BamHl-BcoRl
restriction fragment from pRGC31 in which the overlapping DNA ends
were filled in with DNA polymerase I and then blunt end ligated into
the Smal ends of pKK232-8 that were dephosphorylated.
Seven carb*,
cm*
the
transformants
contained
the
proper
fragment
in
desired
orientation based on Hindll-Bglll double digests and Hindll digest of
plasmid DNA.
34
pYC620 consists of an agarose gel purified 1.2 Kb BgHI-fcoRl
restriction DNA fragment form pRGC31 in which the overlapping DNA ends
was filled in with DNA polymerase I and then blunt end ligated into
the
Smal
ends
of
pKK232-8
that
was
dephosphorylated.
Plasmid
transformants were verified to contain the proper DNA fragment based
on Windlll digestions.
XI.
Purification of FtsA-LacZ hybrid protein.
The
purification
galactosidase
of
affinity
FtsA-LacZ
columns
as
fusion
protein
described
by
was
by
Bastia(47)
Pand
Ullmann(121) with some modifications.
E. coli cells AMC290/pYC3 were grown in YET broth at 30°C in the
presence and carb(25 yg/ml).
The X pL promoter of pYC3 was induced by
heating to 43°C when the culture reach an 0DfeOo of 0.3.
After 2.5 hrs
at 43°C the cells were harvested by centrifugation at 6000 g for 5
min. Pellet was suspended so that a gram of cells was in 2 mis of
buffer I (0.2M Tris-HCl, pH 7.0/ 0.25M NaCl/O.OlM magnesium acetate/
10 mM 2-mercaptoethanol/ 5% glycerol/ ImM PMSF).
was
frozen
at
-80°C
and
thawed
quickly
in
This cell suspension
a
30°C
water
Pancreatic DNase and RNase was added at 10 yg/ml each.
bath.
The cell
suspension was then disrupted by two passages at 10,000 psi through a
pre-chilled French pressure cell.
Unbroken cells were
centrifugation for 10 min. at 2000 X g.
centrifugation at
rotor(Beckman).
35,000
removed by
The lysate was clarified by
rpm for 30 min at 4°C in a type
75 Ti
The P-galactosidase activity in the cleared lysate
was determined as described(83).
35
The
protein
ammonium sulfate.
stirring
to
40%
from
above
cleared
lysate
was
salted
out
with
Ammonium sulfate crystals were added slowly with
saturation.
The
precipitate
was
collected
by
centrifugation and the pellet was suspended in buffer II (lOmM Tris,
pH 7.0/ lOmM magnesium acetate/ 0.2M NaCl/ 0.1M KC1/ O.lmM EDTA/ ImM
DTE/ O.lmM PMSF) and dialysed against 100 vol of the same buffer for 3
hrs with 3 buffer changes.
The above dialysate was mixed with 5M NaCl to obtain a final
concentration of 1.5M NaCl. A volume of 2 ml of this solution was
applied
to
2
ml
bed
volume
of
galactopyranoside-agarose(NH2BenSGal-Agarose)
p-aminobenzyl-8-D-thiocolumn
which
was
equilibrated with buffer III (lOmM Tris, pH7.0/ 1.5 M NaCl/ 0.1M KC1/
0.1M EDTA/ ImM DTE/ O.lmM PMSF).
The flow rate was 1 ml/hr.
The
column was washed with buffer III (about 100 bed volumes) until no
more material absorbing at 280nm appeared in the flow through.
The
protein retained was eluted from the column with 0.1M sodium borate,
lOmM
3-mercaptoethanol,
pH
10,
precipitated with ammonium sulfate.
and
the
proteins
was
promptly
This precipitate was collected by
centrifugation and the protein was resuspended in buffer IV (40mM
Tris-HCl,pH 7.5/ImM MgCl2/0.ImM EDTA/ImM DTE/50% glycerol) and kept at
-80°C.
Before injection into rabbits the protein was dialysed against
phosphate buffered saline(PBS;
lOmM phosphate buffer/150mM NaCl, pH
7.4).
XII.
Production of FtsZ-LacZ Antibodies
Antiserum against
the
FtsA-LacZ
fusion protein
using a immunization protocol previously described(98).
was
produced
A rabbit was
36
injected intradermally with antigen(500 yg) emulsified in an equal
volume of complete Freund adjuvant(Difco).
After 4 week, the rabbit
was given a booster injection of another 500 yg of antigen in same
volume with incomplete Freund adjuvant.
To insure high titer of FtsA
specific antibodies, the rabbit was given a second booster injection
with 200 yg of FtsA-LacZ hybrid protein isolated from SDS-PAGE gels.
Strips of acrylamide excised from gels which had been stained and
dried were rehydrated in PBS buffer and macerated in a mortar and
emulsified in an equal volume of incomplete adjuvant.
Blood was obtained before
experiments (pre-immune serum).
was
titered
at
1
week
the
initial
injection
for control
Antibody production against FtsA-LacZ
intervals
after
the
booster
injection.
Detection and titer of the blood antiserum were carried out by ring
precipitin and dot blotting tests
(17) with aconstant dilution
of
antigen. Pure immunoglobulin,IgG antibody fraction, was prepared from
the
serum
by
chromatography
on
protein
according to manufactures procedure.
A-sepharose
C1-4B
column
Affinity purified rabbit IgG was
dialysed against PBS buffer containing0.02% sodium azide overnight
with
four
changes
immunodetection,
of
buffer.
Toenhance
the
specificity of
the IgG antibodies were absorbed against a cleared
lysate of YC100 (ftsAlO mutant) prepared after a temperature shifted
to 42°C for 2 hrs.
XIII.
Western blot analysis.
Polypeptides separated on a 10 to 30% gradient SDS-PAGE gel were
electrophoretically
transferred
to
a Nytran Sheet
(Schleicher &
37
Schuell) at 250 mA for 3 hrs by the method of
Burnette(17) with the
following modifications.
The transfer buffer consisted of 20 mM Tris-Cl, 150 mM Glycine,
pH
8.0.
Transfer
standards (BRL).
buffer
was
monitored
by
prestained
molecular
weight
The immobilized polypeptides were blocked in TBST
(0.1M Tris-Cl,pH
7.5,
0.1M NaCl,
0.05%
v/v
Triton
X-100)
containing 10 % (w/v) bovine serum albumin(BSA) overnight at 48°C.
The
blocked
membrane
filter
was
then
incubated
with
appropriate
dilutions of antibodies (1:1000) in TBST buffer containing 0.5% BSA
for 2 to 4 hrs.
each),
After being washed in six changes of buffer (10 min
the membrane sheets were
phosphatase
incubated for
linked-goat anti-rabbit
IgG
1 hr with alkaline
(1:7500 dilution)
and then
washed as described above. All the above steps were performed with
gentle
agitation
at
room
temperature
throughout
unless
otherwise
noted.
Antibody reactivity was detected with color development by the
substrates BCIP and NBT. If labelled proteins were used, the resulting
immunoblot was subjected to autoradiography after being dried.
XIV. Cell fractionation procedure.
Membrane, cytosol and Sarkosyl insoluble membrane fractions were
prepared as described previously
(44,101).
Ten mis of cells or
labelled maxicells with plasmids were pelleted by centrifugation at
12,000 X g for 10 min, suspended in 5 ml of 10 mM phosphate buffer, pH
7.4,
centrifuged again, and finally resuspended in 2 mis of the same
buffer.
The cells were then lysed by sonic oscillation.
The un-lysed
cells were removed by centrifugation at 2000 X g for 10 min. This
38
cleared lysate was fractionated into cytosol and membrane fraction by
centrifugation at 20,000 X g for 15 min.
The envelope-containing
pellet was then suspended in 2 mis of buffer.
envelope pellet
preparation was
further fractionated into Sarkosyl
insoluble outer membrane by being
37°C for 20 min.
One half(lml) of the
solubilized with 1% Sarkosyl at
The total membrane and Sarkosyl-insoluble membrane
preparations were centrifuged again at 20,000 X g for 15 min and
resuspended in phosphate buffer.
were
concentrated
by precipitation
The soluble cytoplasmic proteins
with
10%
trichloroacetic
acid,
washed twice with 80% cold acetone and resuspended in smaller volume
of phosphate buffer.
Proteins from the above fractions were separated by PAGE-SDS
gels, and in case of labelled proteins from maxicells were detected by
autoradiography.
XV.
Isolation of membrane fractions 0MH , 0ML , IM.
Cell fractionation of membrane envelope into 0M„, OMt,, IM was
performed by the procedure of Ishidate et al (59) as briefly described
below.
Cells were grown at 30°C in YET medium (1 1) to ODeoo31 0.4,
rapidly cooled and then centrifuged at 2000 X g for 10 min. Pellet was
resuspended in 10 mis of 20% sucrose and
(10 yg/ml each) were added.
pancreatic DNase and RNase
The cell suspension was then mechanically
disrupted by two passages at 1000 psi through a pre-chilled French
pressure cell. (American Instrument Co., Silver Spring, MD.).
After
removal of unbroken cells by centrifugation for 30 min at 2000 X g,
EDTA was added to the supernatant fraction to a final concentration of
39
5
mM.
The
supernatant (1.7
ml)
was
layered
onto
a
two
step
gradient(SGO) consisting of 0.8 ml of 60% sucrose and 2.5m of 25%
sucrose.
The
crudemembrane
fraction,
isolated
from
SGO
by
centrifugation in a Beckman SW50.1 rotor at 40K rpm for 3.5 hrs, was
diluted with lOmM Hepes,
1.365-1.37
at
20°C.
applied to the
(0.5ml),
55%
pH7.4,
The
top of
(0.8ml),
5mM EDTA to a refractive index of
resulting suspension
(2.0 ml)
was
the following sucrose gradient (SGI)
50%
(2.2ml),
45%
(2.2ml),
40%
then
:
60%
(2.2ml),
35%
(1.4ml), 30% (1,0ml). The gradient was centrifuged in a Beckman SW41
rotor at 36K rpm for 16 hrs.
Fractions (0.3 to 0.4ml) were collected
from the top using a fraction recovery system (Beckman) and refractive
index was determined with a refractometer (Bausch & Lomb).
the
refractive
concentrated by
index,
each
membrane
fraction
Based on
was pooled
and
centrifugation at 50K rpm for 3.5 hrs after being
diluted with 3 volumes of 10 mM HEPES buffer(pH 7.4), and the final
pellets were resuspended in lOmM HEPES buffer.
Total
cytosol
centrifugation
of
and
the
membrane
supernatant
fractions
(3
ml)
were
prepared
obtained
above
by
by
centrifugation at 100,000 X g for 1.5 hr after dilution with 2 volumes
of 10 mM HEPES, pH 7.4.
Pelleted membrane was resuspended in same
buffer and soluble proteins were concentrated by precipitation with
10% cold trichloracetic acid(TCA).
The TCA precipitate was allowed to
stand on ice for 20 min, pelleted by centrifugation, washed twice with
80% acetone, and finally resuspended in same buffer.
Approximately equal amounts of protein from each sample (50 pg)
were loaded onto SDS-PAGE gels used for protein separation and Western
immunoblotting as described above.
40
XVI Immunogold labelling and electron microscopy.
Immunogold labelling was performed as described by Bayer et
al.(6)
and by Reid et al.(93) with following modifications.
Cells ( AMC290) were grown in YET broth at 30°C to mid-log phase
and a portion of cells were then fixed with an equal volume of L broth
with 4% formaldehyde and 0,2 M cacodylate-HCl buffer(CD), pH 7.2 for
30 min. The fixed cells were collected on a filter by drawing about 2
mis of the suspension of fixed cells into a 5 ml syringe and passing
through
an attachable
filter holder
containing
a
13 mm
diameter
polycarbonate membrane filter (0.40 urn). The filter was washed with 3
ml
of
0.1M
CD
buffer
and
then
dehydrated
with
graded
alcohol
series(50% to 100%). Dehydration was started in 50% ethyl alcohol.
10% increases of alcohol concentration were used in the dehydration
steps.
The filter membrane was removed from the holder, infilterated
in LR write resin at room temperatures for 1 hr (two changes of LR
white resis) and embedded in fresh accelerated LR white resin in 30
min. on ice.
For gold-labelling, ultrathin sections were placed on
and carbon coated copper grids.
parlodion
These grids were blocked with TBST/5%
BSA buffer (pH 7.5) for 30 min to saturate nonspecific protein binding
sites and then reacted for 1.5 hr with affinity purified FtsA specific
antibodies (1 pg/ul) in dilutions of 1:50 in TBST/ 1% BSA buffer.
In
parallel, sample grids were processed with preimmune IgG antibodies
(0.5 yg/ml) in dilutions of 1:25 as controls.
After five subsequent
washings in TBST/1% BSA buffer (5 min each), the sections were floated
on goat anti-rabbit IgG 10 nm gold conjugate (Janssen's) in TBST/1%
41
BSA buffer.
The grids were again washed 5 times in TBST/1% BSA buffer
and rinsed in distilled water.
Sections were stained in 1% uranyl acetate before being examined
with a JEOL 100CX transmission electron microscope at 80 KV.
Ultra
thin
sections
of
IPTG
induced
cells
(YC200)
were
immunogold-labelled with FtsA-LacZ antibodies as described above.
XVII.
Preparation of cell extracts for enzyme assays.
Cell extracts for CAT/fl-lactamase assays were prepared by the
method of Lupski et al (72).
Cells containing plasmid were inoculated
in L broth supplemented with 0.2% glucose and ampicillin(50 yg/ml) or
carbenicillin(25 pg/ml).
An overnight culture was diluted 100 fold in
fresh broth and grown to an 0D6Oo = 0.2 to 0.5 (Spectronic 20, Baush
and Lomb).
Six mis of cells were collected in a 15 ml corex tube by
centrifugation at 7000 rpm for 10 min in Sorvell SS34 rotor.
The
cells were washed in 2 ml of 50 mM Tris, 30 pM DTT,pH 7.8, resuspended
in 600 yl of same buffer and transferred to 1.5 ml Eppendorf tubes.
The cells were frozen by placing at -70°C for 1 hr.
Cells were then
thawed and sonicated with 2 or 3 pulses of 10 sec with at least 15 sec
rest between pulses using a MSE sonicator with a micro-tip. To remove
cell
debris,
the
cell
extracts
centrifuge at 4®C for 15 min.
were
centrifuged
in
an
Eppendorf
The supernatant was placed in another
Eppendorf tube and kept on ice for subsequent enzyme assays or at 20°C for storage.
42
XVIII Enzyme assays.
a.
3-galactosidase assay
B-galactosidase
activity
was
determined
(ONPG)
Miller(83).
An aliquot of the sample to be assayed was
(final
NaH2POA-H20,
volume
1 ml)
according
containing
to
o-nitrophenyl-
galactoside
buffer
as a substrate
using
the
method
added to Z
60 mM Na2HPOA-7H20,
40 mM
10 mM KC1, ImM MgSO*, 50 mM 8-mercaptoethanol, pH
If the sample consisted
of intact cells, 40 ul CHC13 and
SDS were added and vortex for 10 secs.
of
20 ul
7.0.
0.1%
Sample tubes are incubated in
a water bath at 28°C for 5 minutes for equilibration.
The reaction
was started by adding 0.2 ml of 0NPG(4 mg/ml) and stopped by adding
0.5
ml
of
developed.
was
IM
Na2C03 solution
after
sufficient
yellow
color
had
The optical density at both 420nm and 550nm for each tube
determined
and
the
units
of
P-galactosidase
activity
was
calculated from the following formula:
units = 1000 X (0D^2o " 1.75 X 0Dsso)/t X v X 0D&oo«
Where t is length of reaction time in minutes and v is the volume of
the enzyme extract used per ml of assay mixture.
b . CAT Assay.
Cell extracts as described in section XVII were used in the CAT
assay.
The reaction mixture was freshly prepared by dissolving 8 mg
of DTNB (5,5'-dithiobis-2-nitro benzoic acid) in 2.0ml 1.0 M Tris-HCl,
pH 7.8, adding 0.4 ml acetyl-CoA (5 mM) stock solution, and adjusting
the total volume to 20 mis.
600 yl of the reaction mixture was placed
in a reference cuvette and sample cuvette in a Beckman double beam
recording Model 30 spectrophotometer.
20 yl of enzyme extract was
43
added to thereaction mixture and
measured
for 30 sec to 3 min using the spectrophotometer recorder to
obtain the background activity.
chloramphenicol
recorded
the A«l2 was adjusted to zero, then
for
To start the reaction, 12 yl of 5 mM
was added.
The
increased
absorbance
about 5 min.
The
CAT enzyme
at A* j.2 was
specific activity (in
nmol/min/mg) was determined by dividing nmol/min-ml by the protein
concentration(mg/ml) as follows:
nmoles/min/mg = AA*i 2 X 1/0.0136 X 1/mg protein
C.
fl-lactamase Assay.
Cell extracts as described in section XVII were used.
600 yl of
a reaction mixture consisting of 41.5 yg/ml cephaloridine (10~*M) in
0.1M
phosphate
buffer
pH
7.0
was
placed
in
a
sample
cuvette.
Spectrophotometer was blanked against 0.1M phosphate buffer pH 7.0 and
the A 2 5 5 was recorded for 30 sec. to get a base-line level.
The
absorbance of the cephaloridine mixture (A25s) was between 1.2 and
1.4.
To start the reaction 20 yl of the enzyme extract was added to
the reaction mixture and the A25s was recorded for approximately 5
min.
A fall of the optical density from 1.4 to 0.6 would indicate
complete
hydrolysis.
Specific
nmole/min/mg protein) was calculated
activity
for
B-lactamase
(in
by (AA2Ss/min) X 375/mg protein.
An alternate substrate used was PADAC. Again a 600 yl reaction
mixture was used consisting of 15.6 yg/ml PADAC (2.8 X 10_5M) in 0.05
M phosphate buffer, pH 7.0.
The Specific
activity
PADAC hydrolysis was measured at As&&.
for P-lactamase
in nmole/min/mg protein was
determined by (AAssa/min) X (1/0.0605/mg protein).
44
XIX.
Protein Assay.
Amount of protein was measured by the method of Bradford(15)
using the Biorad Protein Assay dye reagent concentrate. Bovine serum
albumin
(Sigma)
was
diluted
and
used
for
protein
standard
concentrations.
XX. Plasmolysis of bacterial cells
Plasmolysis of bacterial
cells were
method of MacAlister and Rothfield(128).
performed by a modified
A strain AMC410(ftsZ84)
grown in YET broth overnight at 30°C were diluted (1:100) in fresh
medium.
A portion of the growing cells at 30°C (0D6Oo = 0.2) were
shifted to 42°C for 90 min.
in a microfuge for 1 min.
Culture was harvested by centrifugation
The pellet was suspended in 0.1 volume of
fresh culture medium at room temperature.
suspension was
plasmolyzed
by adding
equal
A portion of this cell
volume
of plasmolyzing
solution (26% suerose(wt/vol), 0.2 M cacodylate-Cl buffer, pH 7.2).
These cells were kept at room temperature for 3 min before they were
fixed
by
the
addition
plasmolyzing buffer.
of
equal
volume
of
4%
formaldehyde
in
The cell suspension was allowed to stand for an
additional 60 min. at room temperature, before being centrifuged for
30
sec
in
microfuge
and
being
suspended
in
fresh
plasmolyzing
solution.
Unplasmolyzed cells were also fixed with formaldehyde in
parallel.
Micrographs were taken with a phase contrast microscope
(BH-2; Olympus, Splan lOOpl objective) with a 35 mm camera.
Portion
of the fixed plasmolyzed and unplasmolyzed cells were thin sectioned
and immunogold-labelled as described in section XVI.
RESULTS
I.
Isolation of FtsA-LacZ protein fusions.
pRGC31
plasmids
with
mini-Mu,
MudII1734,
insertions
were
selected as Mu-ductants that were both amp*- and kanr and grew as light
blue colonies on X-gal indicator plates. Approximately 1% of the total
kanr colonies expressed B-galactosidase indicating an in-frame protein
fusion with lacZ.
Of these approximately one in fifty(2%) were found
to be within the ftsA gene by restriction enzyme mapping. The mini-Mu
insertion sites of four isolated ftsA-lacZ
6
550
plasmids are shown in Fig.
In plasmid pYC40, MudII1734 transposon was inserted approximately
bases
from
the
start
of
the
ftsA
amino-terminal
end.
In
plasmid,pYC39, the transposon insertion was approximately 650 bases,
and in plasmids, pYC28 and PYC16, the mini-Mu was inserted 850 bases
from amino-terminal end.
Temperature
sensitive
ftsA
mutant
strains,
TKF10
or
YC100,
remained temperature sensitive at the nonpermissive temperature(42°C)
when the ftsA-lacZ fusion plasmids were introduced.
fusions may
plasmids,
became
retain some
pYC39
and pYC40,
filamentous
filaments
were
functional
at
observed
the
were
larger protein
transformed
permissive
with
the
transformed with pYC39 and pYC40.
slightly
interactions.
fusions,
septation in AMC290.
45
wild
When
the
the
fusion
into strain YC100
temperature
type
However,
(30°C).
strain,
AMC290,
it
Some
when
Interestingly, plasmids encoding
pYC16 and pYC28,
did not
effect
46
II.
Expression of FtsA-LacZ fusion polypeptide in maxicells.
The synthesis
of FtsA-LacZ
fusion proteins was confirmed by
labelling the plasmid specified proteins using the maxicell labelling
procedure(99,100).
Autoradiograms
of
the
[35S]methionine
labelled
polypeptides encoded by several plasmids in strain CSR603 is shown in
Fig. 7.
On this gel system, the FtsA protein migrates as a 48 kD
polypeptide, FtsZ as a 45 kD polypeptide and 8-lactamase as a 28 kD
polypeptide(Fig. 7, lane a, pRG31). All transformants with presumed
ftsA-lacZ fusion containing plasmids synthesized
large polypeptides
migrating near the top of the SDS-PAGE gel with concomitant loss of
the
FtsA
polypeptide
band(Fig.7,
lanesb,c,d).
specified a new 142 kD fusion peptide.
encoded 154 kD fusion
peptides.
Plasmid
pYC40
Plasmids, pYC28 and pYC16
Plasmid pYC39 specified a
hybrid
peptide of 146 kD (data not shown).
III.
Cellular localization of the FtsA-LacZ protein.
It was imperative to determine whether the amino-terminal end of
the
FtsA
protein could
localize
similar to the FtsA protein.
the
FtsA-LacZ
fusion
polypeptide
Native FtsA protein was labelled in
maxicells containing pRGC31(Fig. 7). CSR603 strains containing pYC28,
pYC40 or a control plasmid pRGC43 which encodes P-galactosidase and
FtsA were also labelled.
The labelled maxicells were fractionated
into membrane and cytoplasmic fractions by centrifugation and part of
the membrane fraction was further extracted with 1% Sarkosyl detergent
to solubilize
the inner membrane proteins.
Table
3 presents the
quantitative data as relative molar yields of labelled proteins from
an
electrophoresis
autoradiogram
.
The
8-galactosidase
peptide
47
specified by pRGC43 was localized in the cytoplasmic fraction with
only a small contaminating amount visible in the membrane fractions.
The FtsA protein, specified by both pRGC43 and pRGC31, fractionated
almost equally between the cytoplasmic fraction and the total membrane
fraction.
FtsA
Furthermore, approximately 50% of the membrane fraction of
remained
after
Sarkosyl
extraction.
The
FtsA-LacZ
fusion
proteins specified by plasmids pYC28 and pYC40 behaved identically to
the FtsA protein.
The
presence of FtsA and FtsA-LacZ
labelled
proteins being found
in both the soluble and membrane fractions is
most likely a consequence of the maxicell protein labelling procedure.
Labelled
proteins
are
likely
to
be
synthesized
in
excess,
and
maxicells are division arrested due to extreme chromosome DNA damage.
Since the FtsA-LacZ fusion protein has P-galactosidase activity,
this activity could be used to localize the FtsA-LacZ protein in wild
type cells. We fractionated the membranes of strain AMC290 containing
fusion
plasmids
by
the
procedure
of
Ishidate
et
al.(59).
This
procedure enables the isolation of unique membrane fraction called
"outer
membrane-light. (0Ml)
membrane
fraction.
The
which
contains
enzyme
collected(
activity
in
Fig.
inner-murein-outer
P-galactosidase activity
isopycnic sucrose gradient was determined
fractions
the
8).
membrane
Most
for the
(80%)
from
the
final
different density
of the (5-galactosidase
preparation of the
FtsA-LacZ
fusion
specified by pYC40 was found in the two fractions corresponding to
0M„(density 1.24 to 1.26) and 0ML(density 1.21-1.23). A small amount
of LacZ specified P-galactosidase activity was found in the inner
48
membrane(IM)
1.12).
fraction and slightly lighter fraction(density 1.16 to
Similar results were obtained with cells containing fusion
plasmid pYC16.
IV.
Overproduction and purification of FtsA-LacZ protein.
In order to produce large quantities of the FtsA-LacZ hybrid
protein, the 8.5 Kb BamHI fragment of plasmid pYC16(23) encoding the
hybrid protein was subcloned into the expression plasmid pGW7(30).
Plasmid
pYC16
was
partially
digested
with
BamHl
and
the
8.5
Kb
fragment was subsequently recovered from a preparative agarose gel and
ligated into BamHl digested and dephosphorylated pGW7 DNA.
One kan^
carbr colony contained a plasmid, designated pYC3, which had the 8.5
Kb insert
in the correct orientation as determined by restriction
enzyme digestion with BgllK Fig.9).
When
strain
AMC290
shifted from 30°C to 42°C,
(Alac)
containing
pYC3
are
temperature
transcription of the ftsk-lacZ DNA was
induced resulting in increased fusion protein production. The level of
hybrid protein production was measured by P-galactosidase specific
activity. The optimal P-galactosidase activity was found after 2.5 hr
at
42°C.
These temperature
accumulated up
shifted
pYC3
containing
cells
to 1% of the total cell protein as FtsA-LacZ protein.
P-galactosidase activity was approximately 10 fold higher (7230 U/mg)
after
induction
containing the
(687
U/ml)
and
20
fold
higher
than
same
cells
original fusion plasmid pYC16(347 U/mg).
The hybrid protein was purified from the soluble fraction of
disrupted cells by two purification step: 40% (NH^)2S0A precipitation
cut
and
affinity
chromatography
on
p-aminobenzyl-8-D-thio-
49
galactopyranoside-agarose.
In Fig. 10, the composition and degree of
purity of the total soluble proteins, the 40% (NH«)2S0A fraction and
the affinity-purified fractions separated by SDS-PAGE are shown.
The
40% ammonium sulfate precipitant fraction retained approximately 90%
of total 0-galactosidase activity and there was an enrichment of the
hybrid protein (Fig. 10, lane D).
The hybrid protein had the expected
size compared to P-galactosidase molecular weight marker (Fig.10, lane
E),and was
identical to fusion protein specified by plasmid pYC16
(which had been labelled by maxicell labelling).
polypeptide band,
that was
probably
A 0-galactosidase
a degradation product
of
the
fusion protein, was always present in the affinity-purified protein
fraction.
This protein degradation may be due to enzymatic activity
occurring
during
temperature
affinity
the
purification
which
was
performed
at
room
(to increase the binding of the fusion protein to the
matrix).
The
cleavage
of
fusion
to
P-galactosidase
polypeptide can be expected because FtsA-LacZ fused protein is likely
to have two distinct protein domains.
V.
Identification of wild type FtsA protein by Western blots.
Polyclonal antibodies from rabbit against the FtsA-LacZ fusion
protein antigen were prepared.
The antiserum contained a significant
titer of antibodies to both the FtsA-LacZ and LacZ determined by the
ring preciptin
tests and
by dot
blot
analysis
(data not
shown).
Western immunoblotting experiments were performed with an FtsA-LacZ
immunoglobulin fraction (a final protein concentration of 1 mg/ml that
was purified from serum using a protein A affinity column).
50
To test wild type FtsA protein cross reactivity with the FtsALacZ antibodies, the FtsA protein was [35S]methionine labelled using
maxicell
containing
immunoblotting
was
plasmids
encoding
performed.
FtsA
before
[3sS]methionine
Western
labelled
FtsA
polypeptides, visualized by autoradiography, was also found by Western
blotting to be enzymatically
11B).
labelled by FtsA-LacZ antibodies(Fig.
As expected, both [3sS]methionine labeled fusion polypeptides
specified by plasmids pYC16, PYC40 (Fig.llD, lane 3 & 4) and labeled
0-galactosidase specified by control plasmid pRGC43 also cross reacted
with FtsA-LacZ
antibodies
(Fig.
11D,
lane2).
The FtsA-LacZ
fusion
proteins synthesized in maxicell appeared to be unstable because a
strong antibody cross-reactivity band similar in mobility
to
0-
galactosidase was observed upon Western blotting.'
Thus, the results
indicated
FtsA-LacZ
that
the
polyclonal
antibodies
from
protein
antigen was specific for FtsA, FtsA-LacZ and LacZ polypeptides.
Additional
immunoreactive
bands which stained
less
intensely
(Fig. 1IB, lane 1 & 2) were visible in total cell extracts. One at 37
Kdal and several small peptides at the bottom of the Western blot.
These
bands,however,
were
not
observed
with
the
soluble
protein
fraction probed by Western blotting with FtsA-LacZ antibodies.
That
is, these extraneous bands appeared in only the membrane fractions.
These bands could be the result of cross reactivity of FtsA antibodies
with other membrane proteins, modified FtsA protein or its breakdown
products.
Nonspecific
binding
could
not
be
completely
excluded
because preabsorbtion to remove nonspecific antibodies were performed
with a cleared lysate.
However,immunoglobulins from
pre-immune serum
51
did not show any reactivity in
Western blots of membrane proteins
under the same experimental conditions(data not shown).
To determine whether the FtsA protein synthesized in wild type
cell could be detected with the FtsA-LacZ antibody preparation,total
cellular polypeptides from a Lac deletion strain AMC290 were separated
on SDS-PAGE and Western blotted with several concentration of FtsALacZ antibodies (data not shown).
The amount of FtsA within a wild
type cell was apparently too small to be detected in a total cell
extract.
Wild type FtsA protein was detected in an immunoblot of
protein from a 40% ammonium sulfate pelleted fraction of AMC290/pYC3
which was
temperature
peptide (Fig. 12).
shifted to overproduce
the FtsA-LacZ
fusion
Apparently, this plasmid containing strain either
synthesized more FtsA or wild type FtsA became more stable because of
the FtsA-LacZ fusion protein synthesis. However, this experiment gave
us confidence that we could detect wild type FtsA if a cell fraction
was enriched for FtsA.
VI Cellular localization of FtsA protein.
Membrane fractionation studies with FtsA-LacZ expressing strains
revealed that 0-galactosidase activity was associated with membrane
fractions
corresponding
membrane light (OM*,).
to
outer
membrane
heavy(0M„)
and
outer
Previous studies above demonstrated that we can
detect FtsA protein by Western blotting.
Thus,to verify that native
FtsA protein indeed was enriched and localized to membranes in wild
type E. coli cells two experimental approaches using the FtsA specific
polyclonal antibodies were performed: A. Western immunoblots of cell
fractions and B. Immunoelectron microscopic localization.
52
A.
Western blots of cell fractions.
AMC290(Alac74) cells were fractionated into cytosol and envelope
membrane
fractions
first
by centrifugation.
fraction were separated by SDS-PAGE,
The protein from both
blotted onto Nytran membrane
filters and probed with FtsA-LacZ antibodies.
A band in the envelope
fraction, corresponding in molecular weight to native FtsA, interacted
with FtsA-LacZ antibodies but not in the soluble fraction (Fig.13,
lanes 5,6,8,9).
Increased g-mercaptoethanol concentration from 1 to 5% in PAGE
buffer in attempts to remove the cross antigenic smearing that was
found with SDS-PAGE of the envelope fraction( Fig 13) resulted in the
labelling of two bands (60Kdal and 52Kdal) above the 48K FtsA band.
In addition,
another and
band
(37Kdal)
below the FtsA band was
labelled in SDS-PAGE of envelope and soluble fractions(Fig. 14, lane
E,F).
Two bands corresponding to 33Kdal and 23Kdal polypeptides and
one immunoreactive band at the bottom of the gel were also labelled in
SDS-PAGE of the envelope protein fraction. The later two bands, 33Kdal
and
23Kdal,
because
are probably
these
intensively,
as
degradation products
polypeptide
bands
[35S]methionine
are
of
FtsA.
observed,
labelled
This
although
polypeptides
is
less
of
maxicell
into
specific
containing plasmid pRGC31(specifying FtsA).
The
cell
envelope
was
further
fractionated
membrane domains using neither lyzozyme or EDTA but French pressure
cell lysis followed by sucrose floatation steps and isopycnic sucrose
gradients as described previously (section III).
0MH , 0ML and IM
membrane fractions were separated on SDS-PAGE(10-30%) and the proteins
53
were electro-transferred to Nytran sheet.
result of
Fig. 13 and 14, show the
Western blotting with FtsA-LacZ antibodies.
In Fig. 13
(lanes 1,2,3,4) the data revealed that native FtsA cross reactivity to
FtsA-LacZ antibodies were found in QM„ and 0ML fraction of AMC290
(0D6OO=1.6).
Thisresult
studies.
is consistent with the FtsA-LacZ
Similarresults
immunoblotting membrane
were
obtained
after
fractions of YClOO(ftsAlO)
shifted to 42°C for 2 hrs.
fusion
Western
strain that was
This indicated that ftsAlO does make a
cross reactive protein which is incorporated into the envelope but
probably is not functional.
When
buffer,
fraction
B-mercaptoethanol
the
cross
but
also
concentration
reactivity
in
IM
to
FtsA was
fraction
was
found
(Fig.
14,
increased
not
in
only
lane
PAGE
in 0ML
A,B,C,D)
of
AMC290(0D6Oo=0.4). The labelling of the cross reacting peptides were
due
to
antibody specificity
conjugant labelling because
immunoblotting
and not due
to any spurious
enzyme-
similar results were obtained when Western
was performed with a different secondary antiserum,
125I-labelled goat anti-rabbit IgG under same experimental conditions.
Fig 15 presents the SDS-PAGE profile of Coomassie blue stained
polypeptides which Western blotted and presented in Fig. 14.
The
membrane
fractions
additional immunoreactive
(Fig.14,lanes
To
exclude
the
contained
labelled bands similar to labelled bands
observed in total membrane fraction and the
E,F).
A,B,C,D)
possibility
soluble fraction
thatthe
appearance
of
(Fig 14,
these
additional bands was due to nonspecific binding (for example, cross
reactivity to the major outer membrane protein, OmpA(37Kdal)), FtsALacZ IgG fraction was preabsorbed twice with a lysate containing cell
54
membrane
from
SGO
fraction
nonspecific antibodies.
fraction
is shown
additional
bands
(see Material
Methods)
to
remove
The result of Western blotting with this IgG
in Fig.
and
and
also
16.
of
The
the
labelling
FtsA
band
intensity
of the
decreased.
These
results,although not conclusive, indicated that the several additional
immunoreactive bands are
not
result
cross
reactivity of FtsA-LacZ
antibodies with any major E. coli membrane proteins.
It is more
likely they are precursors, breakdown products or modified forms of
FtsA protein.
The presence of 4M urea in the sample solubilization buffer was
required to cleanly detect the FtsA protein by Western blotting. If
urea was left out of sample buffer, FtsA band was not visible and
cross immunoreactive material migrated near top of gel.
The urea
effect, probably indicates that FtsA aggregates with other membrane
protein or itself even in the presence of 1% SDS. (Increased SDS in
solubilization
buffer
resulted
in
the
inability
of
FtsA-LacZ
antibodies to cross-react with gel proteins.)
B.
Immunogold labelling and electron microscopy.
a. Wild type cells.
Direct
immuno-electron
antibody
using
colloidal
gold
goat
to
microscopic
anti-rabbit
mark
E.
coli
observations
immunoglobulin
cell
bound
of
FtsA
conjugated
with
FtsA
antibody
were
performed. Basically, ultrathin sections of AMC290 cells were isolated
by
low
temperature
embedding
of
cells
in
LR
white
accelerator at 4°C and reacted with FtsA-LacZ antibodies.
resin
with
Binding of
55
these antibodies was detected with goat anti-rabbit IgG conjugated to
gold particles.
In
fig.17(A,B,C,D)
aggregates
in
the
the gold particles were found
envelope
with
few
being
found
in
mostly as
the
central
cytoplasmic region. No gold particles were found in samples treated
withpreimmune
antibodies(Fig.17;
E,F)
instead
of
FtsA-LacZ
antibodies. The gold particles appeared to be aggregated at sites that
appear to be septa or one polar end of the cell.
As
a control,
0-galactosidase
was
induced
with
IPTG
(YC200,lac+) and the cells were thin section and treated as above.
Most
of
the
gold
particles
were
found
throughout
the
cytoplasm
(Fig.17;G,H).
b.
Plasmolyzed cells.
Potential septation sites can be identified by phase contrast
microscopy
and
cells (78).
sites
and
by
electron
microscopy
using
plasmolyzed
bacterial
The resulting plasmolysis bays define potential septal
a
adhesion zone.
cellular
region
consisting
of
inner-outer
membrane
To determine whether inner-outer membrane
regions
within Fts filaments could be visulized, cells were plasmolyzed and
electron
micrographs
of
ultrathin
sections
were
analyzed.
As
described previously, ftsZ84(ts) cells at elevated temperature results
in the formation of long filaments without any visible septa.
When
ftsZ(AMC419) mutant filaments were plasmolyzed, multiple plasmolysis
bays were visible along the length of the filament (Fig. 18).
various sized nonseptal plasmolysis bays appeared at a
The
near cell
length intervals expected if bays define potential septation sites.
56
However, the number and distance of the plasmolysis bays along the
length of the filaments varied somewhat with sucrose concentration and
with growth conditions.
bays were
ftsl(ts)
Similar distribution patterns of plasmolysis
observed by phase
filaments
which
contrast
also
have
microscopy
some
of ftsAlO(ts)
visible
or
constrictions
identifying septal sites(data not shown).
To study distribution of FtsA protein in ftsZ nonconstricting
filamentous cells direct immunoelectron microscopy was performed.
The
electron micrographs (Fig. 19) show that gold particles representing
antibody bound FtsA were found mostly as aggregates at intervals along
the envelope of the nonseptal filaments.
Immunoelectron micrography
of
plasmolyzed
ftsZ
filaments,
to
locate inner-outer membrane regions, were also analyzed under same
experimental conditions.
particles
were
The micrograph in Fig. 20 reveals that gold
distributed
as
aggregates
at
junction sites defined by the plasmolysis bays.
inner-outer
membrane
Furthermore, the gold
particles were clustered at regular intervals along the length of the
filament similar to the non-plasmolysed cells(Fig. 19).
These results
confirm that FtsA proteins are associated with potential septation
sites.
VII. Investigation of ftsZ gene expression.
A.
Promoter gene fusions and quantitation assays.
The vector used in all of the promoter fusion experiments was
pKK232-8
(16).
It
contains
a
chloramphenicol
acetylase(CAT)
structural gene whose natural promoter has been deleted( Fig.
The CAT
21).
gene in this vector can be expressed from a promoter inserted
into an upstream restriction enzymes polylinker site.
Translation
57
proceeds only from a start site immediately flanking the gene.
The
amount of CAT activity in a cell harboring the plasmid is thus an
accurate index of the promoter strength of DNA fragments containing
promoters
which
are
inserted
into
the
polylinker
site.
The
constitutively expressed (J-lactamase gene on the plasmid provides a
selectable marker and an internally expressed gene for normalization.
That is, the ratio of CAT activity to (3-lactamase activity gives a
measure of the strength of the inserted promoter that is independent
of plasmid copy number and overall metabolic activity of the host
cell.
The different ftsZ promoters (PziPzaPza) and ftsA (pA) promoter
regions were subcloned into pKK232-8 polylinker region and then CAT/fJlactamse specific activity in wild type strain (AMC290) background
(Table 4) were determined.
The potential proximal promoters(110), Pzx
and Pz2, within the Hindlll-EcoRl DNA fragment of the plasmid pYC580
was poorly expressed in vivo being approximately 21.8 X 10-3 units.
This is consistent with low level of ftsZ expression from a AenvA(77),
that only partial complemented a ftsZ84(ts) mutant.
In contrast to
PziPza. the promoter Pz3 within the coding sequence of ftsA, as in
PYC530, showed a high level of promoter activity, approximately 235.2
X 10-3 units.
If the' ftsA gene promoter, PA , was fused to Pz3 (BamHl-
Bglll), as in pYC500, then the promoter activity increases slightly
indicating a weak ftsA. promoter. This indicates that the ftsk promoter
is weak and contributes little to the transcription from Pa Pz3-CAT
fusion.
The total promoter activity from pYC620 was lower than the
sum of the separate
included with
activities of
PzaPzaPzi.
as
Pz3 and PziPz2 -
in pYC600,
the promoter
When PA was
activity was
58
increased by 1.8 fold but still lower than the level of transcription
from pYC53(P23).
Lutkenhaus and his colleagues!.'75) have shown that the presence
of
part
of
ftsQ
structural
gene,
as
in
pYC600,
results
in
the
decreased ftsZ expression and TnlO insertion into ftsQ gene recovers
ftsZ
gene
expression
enough
to
suppress
the
SOS
induced
lethal
filamentation of the Ion mutant. However, the maximal expression of
ftsZ either with a single copy phage vector (130), or with a multicopy
plasmid(122)
has been shown
to require approximately
2 Kb of DNA
upstream of the ftsZ gene, including ftsQ promoter.
B.
Effects of FtsA, FtsQ, FtsZ, and DnaA on ftsZ expression.
Expression of ftsZ has been cited to be negatively regulated at
the transcriptional level by FtsI, FtsQ, FtsA and FtsZ itself(36).
Temperature
shift experiments with temperature sensitive mutants of
ftsQ, ftsA, and ftsZ were performed to assess whether any ofthe fts
gene products regulate the transcription of another fts cell division
gene.
Since temperature sensitive fts mutants result in an inactive
protein
product
at
the
nonpermissive
temperature,
the
effect
on
transcription is easily determined by measuring the CAT/8-lactamase
specific activities,
using the promoter fusion plasmids constructed
above.
Table 5 shows that FtsZ temperature inactivation increased CAT
expression from both PAPZ3 and Pz3.
1.5
to
3 fold.
Wild
type
The level of increase was about
control
also
activity from PAPZ3 and Pz3 by 3 to 4 fold.
increases
the promoter
Autoregulation of ftsZ
expression can't be completely excluded although the data suggest that
59
the increased CAT expression by FtsZ inactivation could result from
the temperature shift from 30°C to 43°C.
LacZ-transcriptional
studies
which
The data is comparable to
showed
no
apparent
ftsZ
autoregulation (R. Gayda, unpublished).
FtsQ
inactivation
(Table
6) had stimulating
effects both on
PAPz3 promoter combination and on Pz3 promoter alone.
The FtsA inactivation (Table 7) had a slight decreasing effect
on Pz3
promoter with
only
little
compared with wild type control.
effect
on PzaPZi promoter when
A low level of CAT expression was
obtained because of the temperature sensitivity of the ftsA mutant
strain even in the presence of high salt.
These data are opposite to
the finding(36) that transcription of ftsZ is increased as much as 10
fold when cells have a nonsense mutation in the chromosomal copy of
ftsA gene.
On the other hand,
expression
of ftsA
activation
of transcriptionbecause
may
be
there is a evidence supporting that the
controlled
the
by
FtsA
FtsZ
gene
proteinthrough
expression
of
plasmid pRGC43 was no longer sufficient to complement ftsk mutants
when the ftsZ gene was inactivated by the mini-Mu insertion (R. Gayda,
unpublished).
Possible dnak consensus sequence within ftsZ promoters(PzaPzi)
led us to study if dnak effected the expression of ftsZ gene.
Table 8
shows that dnak temperature inactivation increased CAT activity nearly
2 fold from both PAPZ3 and Pz3, suggesting that dnak regulation may
link septation with DNA synthesis.
DISCUSSION
Previous genetic studies have postulated that the function of
FtsA
protein
is
both
regulatory
and
structural
in
septum
formation(115-118).The purpose of this study was to investigate the
structural
involvement
of
FtsA
in
septum
formation.
The
establishment of the cellular location of FtsA is important because
current data (10,29,31,41,65,119) strongly proposed interactions among
the components of the division machinery.
within
a
specific
membrane
domains
Thus the locating FtsA
will
make
possible
the
determination of the specific functions or biochemical activities of
FtsA in septation and will begin to unravel the ordered morphogenic
molecular mechanisms involved in the septation process.
Evidence obtained that FtsA protein is acting as an inner-outer
membrane junction protein are as follows.
First, FtsA-LacZ fusion
proteins demonstrated that the amino terminal end of the FtsA protein
can localize FtsA-LacZ hybrid protein to the cell envelope (Fig. 6 and
Fig.
7).
Furthermore, FtsA-LacZ translational fusions behaved like
FtsA protein when radioactively labelled using the maxicell technique
and
when fractionated into membrane and cytoplasm (Table 3).
Second,
wild type dividing cells synthesizing the FtsA-LacZ hybrid proteins,
when the membranes were fractionated by a modified sucrose equilibrium
gradient
exhibited,
3-galactosidase
corresponding to OM„ and 0ML
(Fig.
inner-outer membrane fusions(59).
were
made
from
purified
experiments
with
these
enzyme
9).
in
fraction
The 0ML fraction contains
Third, when polyclonal antibodies
FtsA-LacZ
antibodies
60
activities
proteins,
on
cell
Western-blotting
membrane
fractions
61
demonstrated that native FtsA was found
fractions
(Fig.
13).
concentration of
localized to 0M„ and 0ML
(Other experiments conducted with
increased
0-mercaptoethanol in SDS-PAGE showed that the FtsA
can also be detected in inner membrane(IM)
fraction
(Fig
15,16)).
Fourth, direct immunoelectron microscopic localization confirmed the
specific localization of FtsA protein at septation sites within the
wild
type
cells.
The
immunogold-labelling
of
thin
sections
of
filamentous cells indicated an enrichment of FtsA protein at potential
septation sites which were identified as inner-outer membrane junction
sites by plasmolysis. Thus, our model is that FtsA along with other
septation proteins forms part of a "septalsome"
and
modulates
the
sequence
of
biochemical
core which initiates
changes
that
lead
to
formation of the division septum.
Ishidate
et
al.
(59)
have
reported
that
the
0ML
membrane
fraction is involved in murein synthesis and the translocation of
newly
synthesized
lipopolysaccharides.
Recently,
MacAlister
et
al.
(78) have demonstrated that 0ML fraction also contains the periseptal
annuli which flank the constriction sites around the potential septum
as an inner-outer membrane gasket to compartmentalize the periplasmic
space between the dividing cells.
Similarly,
it
has
been
reported
(4,97)
that
the
penicillin
binding proteins (PBPs) are located at discrete zones within the cell
envelope, an inter-membrane fraction probably corresponding to innerouter membrane adhesion sites or periseptal annuli(5,78).
PBPs are a
set of actual enzymes that catalyze the insertion of new material into
the peptidoglycan sacculus and are engaged in the control of cell
elongation(106) and cell division(14,63). One of them, PBP3(ftsI) gene
62
product, is a known septation proteins which has transglycosylase and
transpeptidase
enzymatic
synthesis
septum
and
activities
formation.
which
are
involved
Overproduction
of
in
murein
inactive
PBP3
results in inhibition of cell septation implying that PBP3 is part of
a protein complex
in
vivoi15B).
FtsA mutants
have
increased
resistance to 0-lactam induced lysis and decreased binding of [12SI]
ampicillin to PBP3.
FtsA.
an
This supports a possible interaction of PBP3 with
In fact, it has been suggested that FtsA functions as part of
active
multimeric
biosynthesis
for
complex
septum
by
regulating
formation(23).
or
directing
Furthermore,
murein
indirect
evidence, which comes from murein synthesis studies by topographic
autoradiography,
show
that
increased
incorporation
of
radioactive
diaminopimelic acid in the central zone representing the present or
future site for septum formation (127).
Observations(data not shown),
that fusions specified pYC39 or
pYC40 caused some filamentous cell growth even in wild type strains
also supports a functional role of FtsA as a component of multimeric
complex in ^septation.
FtsA-LacZ fusions may be competing with wild
type FtsA protein that may be functioning as an oligomer or mixed
oligomer with other septation proteins.
The
fractions
localization
of
like
FtsA protein indicates
native
FtsAlO
protein
to 0M„ and 0ML membrane
that
FtsAlO
probably
leads to a nonfunctional complex due to an altered protein structure.
FtsZ probably is also involved in the multienzyme complex.
highly
expressed
promoter(124)
death.
LacZ-FtsZ
protein
under
control
of
the
A
lac
when induced by IPTG(123) result in filamentation and
Whereas,
overproduction
of
FtsZ
results
in
early
cell
63
septation and minicell
between
specific
formation(124).
alterations
of
FtsZ
This functional similarity
and
overproduction
of
FtsZ
supports an involvement of some type of stoichiometric regulation of
this complex division machinery.
The ftsA gene consists of 420 residue amino acids(95) with a
calculated molecular weight of 45.4 Kdal.
The FtsA protein has been
identified as a 50 Kdal polypeptide (76) on SDS polyacrylamide gels of
extracts
of
in
vivo
transducing phage.
labelled
cells
with
ftsA
on
plasmid
or
A
This 10% discrepancy of this protein on SDS-PAGE
gels that has been reported (95) is probably due to fact that it is a
membrane protein.
The FtsA gene product in relationship to FtsZ is synthesized
much
less
molecules
(a
of
few
molecules(53)
FtsA
per
cell
of
FtsA
(52,76):
to
maximally
based
on
a
hundred
intensity
of
autoradiograms(76), FtsA is expressed as much as 10 fold less than
FtsZ, and based on the amount of 0-galactosidase enzyme activities of
FtsA
transcriptional
fusions
compared
with
FtsZ
transcriptional
fusions FtsA is expressed as much as 40 fold less than FtsZ.
amount of FtsA protein within
The low
cells could be because FtsA protein is
synthesized at a low level, or because FtsA is degraded at a high
rate.
Our results indicate that both are likely occurring.
Assuming
that the specific activity of native 0-galactosidase is very similar
to that of fused FtsA-LacZ protein activity (where 0.06 units of 0galactosidase activity corresponds
cell(92)).
to 0.3 monomers
of protein per
Assuming that the cell on average contains 50 copies of
fusion plasmid per cell (1 transcript or 1 translation per transcript
per plasmid per generation)
We estimate that there are 20 to 200
64
(probably averaging around 50) FtsA protein molecules per cell from
the
amount
of
P-galactosidase
enzyme
activities
of
FtsA
transcriptional and translational fusions.
Promoter strength
assay
(Table 4)of
ftsA promoter and the
immunoelectron microscopic localization (Fig. 17) are consistent with
this low level expression of ftsA.
containing
fusion
protein
Immunoblot(Fig. 11D) of extracts
specified
by
pYC40
and
enriched
fusion
protein specified by pYC3 also strongly suggest that FtsA protein is
unstable because only g-galactosidase part
of hybrid proteins were
intensely
antibodies
cross-reacted
with
FtsA-LacZ
whereas
no
immunoreactive band corresponding to the FtsA part of fusion protein
was observed.
The difficulty in identifying FtsA protein on immunoblots even
when cells carrying ftsA multicopy plasmid could be ascribed to this
small
amount
of
FtsA
protein.
A 10
fold
increased
antibody
concentration did not increase the sensitivity of FtsA antigenicity in
the immunoblots under same experimental condition.
Interestingly,
the
amount
of the
native
FtsA
protein was
sufficient to be detected in immunoblot (Fig. 12) when 40% (NH*)2POA
precipitate of FtsA-LacZ hybrid proteins enriched cell extracts were
analysed.
This
observation
suggests
that
overproduced
FtA-LacZ
protein could act as a positive regulator of ftsA expression or remove
its negative regulator.
increase
the
stability
Alternatively, FtsA-LacZ hybrid protein could
of
the
native
FtsA
protein
by
a
mixed
multimeric formation or autoregulation.
Conditional
temperature
sensitive
mutants
of
ftsA
form
long
multinucleated non-septated filaments that have slight indentations at
65
the
septation
sites.
This
mutant
phenotype
plus
other
genetic
studies(9,35,76) suggest that FtsA protein acts after the indentation
of
the
new
analysis(113)
septation
of
cell
sites.
Morphogenic
division
mutants
and
have
physiological
proposed
that
the
morphogenetic pathway is in the order of FtsZ-FtsQ-FtsA,FtsI.
Subcloning,
(95,96,109,129,130)
ftsk-ftsZ) form an
promoter
fusionassay,
and
DNA
sequencing
have confirmed that contiguous fts genes (ftsQatypical
operon with overlapping transcriptional
units that transcribe in the same direction from the promoters (Fig.
1) locating upstream of each of the genes.
terminators between the genes.
There are no transcription
This closely packed organization may
imply that the variable levels of coupled expression of these genes
are critical to the ordered functions of cell septation gene products.
The observation that the presence of the ffindll-EcoRl DNA sequences
decreased the ftsZ
expression from
Pz3 by 2 to 4 fold
(Table 4)
suggests that the normal chromosomal location of ftsZ genes for proper
functioning of the
gene is critical
and/or mutual regulation.
to their coordinate
expression
A report(133) that integration of a A phage
that carries a wild type ftsA sequence into the attA site of a ftsk
mutant, can only partially complements this ftsk mutant, point to an
importance of the neighboring DNA sequences.
In vivo quantitative promoter strength assays (Table 4) of the
ftsk and ftsZ promoters in BanMl-EcoRl fragment(2.2Kb) subcloned in
promoter
probe
vector
pKK232-8
relatively weak promoter.
confirms
that
ftsk
promoter
is
a
The potential proximal promoter PZi and Pz2
within the Hindlll-EcoRl fragment was also poorly expressed in vivo
compared
to
the distal
promoter Pz3
in Bglll-Hindlll
region.
A
66
comparison of the homology scores of known promoter sequences -35 and
-10 region to the ftsZ promoter sequences gave the following scores:
PZi= 53.3%, Pz2=45.6% and Pz3 = 37.3%.
limit for an effective promoter.
vivo
data
together
controlled
whereas
suggest
Pz3
that
must
be
A score of 45% is the lower
These homology scores plus our in
PZ1
and
Pz2
positively
may
be
controlled
negatively
to
be
an
effective promoter.
There
is
some
disagreement
of
our
expression
data
with
expression data published by Sullivan and Donachie (109) and by YI and
Lutkenhaus (130).
However, there is also disagreement between these
two published papers.
The differences suggest that this DNA region
may be highly regulated by multiple factors linked to cellular events
and by multiple interactions to control the variable levels of coupled
expression at different times in the cell cycle. The differences are
also likely to be the result of our plasmid constructions and the
interaction of DNA sequences around these promoters.
Expression study
(Table
7) with temperature sensitive mutant
ftsA showed that FtsA appears to increase the ftsZ expression from Pz3
at transcriptional level.
Interestingly, there is an observation that
ftsk expression may be controlled by FtsZ protein through activation
of transcription because the ftsk gene of plasmid pRGC43 is no longer
able to complement an
ftsk mutant when the ftsZ gene was inactivated
by the mini-Mu insertion (R. Gayda, unpublished).
On the other hand,
ftsZ expression(Table8) may be regulated
negatively by
dnak protein
replication.
DnaA protein has been shown to autoregulate its own
synthesis
is an
and
essential
important
control
for initiation of chromosome
element
of
E.
coli origin
67
region(43).
the
Consensus sequences for DnaA binding have been found in
regulatory
regions of
pyrBl, argFl,
polA,
uvrA and dam genes
(111). The promoter expression data plus the DnaA consensus binding
sequence within ftsZ promoters (PZ2PZ1) suggest that dnaA regulation
of ftsZ expression may link septation with DNA synthesis.
Protein sequence of FtsA(95) doesn't contain a typical leader
signal peptide, that is
present in precursors of most periplasmic and
outer membrane proteins.
This indicates that localization of FtsA
into specific inner-outer membrane junction sites may occur through a
different mechanism by which an internal protein sequences of FtsA is
sufficient for proper insertion at a particular cellular location.
The inner-outer membrane fraction is likely to be a labile structure.
The
membrane fractionation procedure employed neither lysozyme or
EDTA treatments in order to preserve envelope structures. Furthermore,
this lability probably explains FtsA-LacZ and FtsA being found in the
0M„ membrane
fraction(Fig.
8
and
13).
This
is
supported
by
the
report(A) that PBPs are localized in the intermembrane fraction under
the mild conditions for cell rupture and usually found in both the IM
and OM membrane fractions when the inner-outer adhesion sites are
destroyed by the cell breakage and fractionation procedures.
Immunoblot
analysis
with
membrane
fractions
isolated
from
sucrose equilibrium gradients indicates that the FtsA is localized to
the specific domain of the envelope,
sites.
60Kdal,
evidence
inner-outer membrane junction
However,immunoreactive bands in addition to FtsA antigen at
52Kdal,
33Kdal,
exclude
the
23Kdal were observed.
possibility
that
Several pieces of
nonspecific
binding
is
68
responsible for the appearance of these immunoreactive bands.
1.
Preimmune serum did not show binding on Western blots under same
experimental conditions.
corresponding
[35S]
2. Two of the bands were also detectable as
labelled bands,
but
specifying plasmid proteins were labelled
Instability
of
FtsA
protein
suggest
less
intensely,
when FtsA
by maxicell procedure.
that
33Kdal
and
23
3.
Kdal
polypeptides are probably FtsA breakdown products or modified FtsA
present in the membrane.
The report that outer membrane protein OmpT
is a protease(49) may support this.
4.
Two times preabsorption of
FtsA-LacZ antibodies fraction with lysate containing total membranes,
to remove antibodies reacting with the major membrane proteins of E.
coll,
decrease
FtsA
antigenicity
but
did
not
eliminate
these
immunoreactive bands.
The mechanisms used by the cell to identify the proper
site and to localize the cell division machinery at this site are
unknown. Rothfied's group(25) has proposed a model that nascent annuli
are generated from a previous periseptal annuli acting as a boundaries
of tseptation site and then laterally displaced till it
reaches a
midpoint between a cell pole and the generating periseptal annuli.
Phase contrast and electron micrographs demonstrate the patterns of
localized plasmolysis bays of periseptal annuli at septation sites and
annular adhesion zones
at nonseptal locations corresponding to future
sites of cell division.
Similar patterns
phenotype
properties
of
localized
in different
plasmolysis
fts
cell
bays
plus
similar
division mutants (.ftsZ,
ftsk, ftsl) ( data not shown) suggest that FtsZ function, leading to
the initiation of septal invagination, appears to be activated after
69
the maturation and localization of nacent periseptal annuli.
The fact
that ftsZ84 filaments after temperature shift back to 30°C, recovers
cell
septation quicker than
model.
However,
the
ftsA or pbpB
possibility
of
filaments supports
FtsZ
protein
is
involved
this
in
localization of the septation sites must also be considered because of
minicell
formation
localization
by
due
to
immunogold
increased
electron
FtsZ
proteins.
microscopy
near
FtsA's
inner-outer
membrane junction sites, when visualized by plasmolysis bays, suggests
another possible role of nascent annuli.
It is probable that the
maturation of
the
periseptal
annuli
mediates
localization
of
the
septation proteins to the proper cell locations.
Thus, our data supports that the septation proteins form a core,
part of an hypothesized "septalsome", a multienzyme-protein complex at
septation sites.
Formation of this multimeric complex is probably
modulated by multiple factors linked to several metabolic processes.
Multiple
interactions
septation proteins
appear to
in the
be
involved
septalsome which
in activation of the
intern
modulates
the
enzymatic activities of the septation steps at precise times during
the cell division cycle.
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115.
Tormo, A., A. Dopazo, A.G. de la Campa, M. Aldea, and M.
Vincente. 1985. Coupling between DNA replication and cell
division mediated by the FtsA protein in Escherichia coli: a
pathway independent of the SOS response,the TER pathway. J.
Bacteriol. 164: 950-953.
116.
Tormo, A., and M. Vicente. 1984. The FtsA gene product
participates in formation of the Escherichia coli septum
structure. J. Bacteriol. 157: 779-784.
117.
Tormo, A., C. Fermandez-Cabrera, and M. Vicente. 1985. The
ftsk gene product: a possible connection between DNA
replication and septation in Escherichia coli. J. Gen.
Microbiol. 131: 239-244.
118.
Tormo, A.,E.R. Martinez-Salas,and M. Vincente. 1980.
Involvement of the ftsk gene product in late stages of the
Escherichia coli cell cycle. J. Bacteriol. 141: 806-813.
119.
Tormo, A., J.A. Ayala, M.A. de Pedro, M. Aldea, and M. Vincente.
1985. Interaction of FtsA and PBP-3 proteins in the
Escherichia coli septum. J. Bacteriol. 166: 985-992.
12 0 .
Utsumi,R., M. Kawamukai, H. Alba, M. Himeno, and T. Komane.
1986. Expression of the adenylate cyclase gene during cell
elongation in Escherichia coli K-12. J. Bacteriol. 168: 14081414.
121.
Ullmann, A. 1984. One-step purification of hybrid proteins
which have B-galactosidase activity. Gene 29: 27-31.
122.
Walker, J.R.,
Regulation
mutants of
formation.
123.
Ward, J.E. and J. Lutkenhaus. 1984. A LacZ-FtsZ gene fusion is
an analog of the cell division inhibitor SulA. J. Bacteriol.
157: 815-820.
124.
Ward, J.E. and J. Lutkenhaus. 1985. Overproduction of FtsZ
induces minicell formation in E. coli. Cell 42: 941-949.
125.
Way, J.C., M.A. Davis, D. Morisato, D.E. Roberts, and N.
Kleckner. 1984. New TnlO derivatives for transposon
mutagenesis and for construction of LacZ operon fusions by
transposition. Gene 32: 369-379.
A. Kovarik, J.S. Allen, and R.A. Gustafson. 1975.
of bacterial cell division: temperature-sensitive
Escherichia coli, that are defective in septum
J. Bacteriol. 123: 673-703.
81
126.
Wientjes, F.B., T.J.M. Olijhoek, U. Schwarz, and N. Nanninga.
1983. Labeling pattern of major penicillin-binding proteins
of Escherichia coli during the division cycle. J. Bacteriol.
153: 1287-1293.
127.
Woldringh, C.L., P. Huls, N. Nanniga, E. Pas, P.E.M. Taschner,
and F. B. Wientjes. 1988 Autoradiographic analysis of
peptidoglycan synthesis in shape and cell division mutants of
Escherichia coli MC4100. In Antibiotic Inhibition of
Bacterial Cell Surface Assembly and Function. (P. Actor et
al., eds) American Society of Microbiology, Washington D.C.
128.
Wolf-Watz, H., and S. Normark. 1976. Evidence for a role of Nacetylmuramyl-L analine amidase in septum separation in
Escherichia coli. J . Bacteriol. 128: 580-586.
129.
Yi, Quing-Ming and J.F. Lutkenhaus. 1985. The nucleotide
sequence of the essential cell division gene ftsZ of
Escherichia coli. Gene 36: 241-247.
130.
Yi, Q.-M, S. Rochenbach, J.E. Ward and J. Lutkenhaus. 1985.
Structure and expresssion the the cell division genes ftsQ,
ftsA, and ftsZ. J. Mol. Biol. 184: 399-412.
Table 1.
Strains
Bacterial Strains
Relevant markers
Derivation,source or
genotype
RGC123
caRS(lon)tsul+
trp,lacAK74, capR9 (45)
RGC103-9
capR9,sulBS
(45)
TKF10
ftsAlO(ts)
(77)
T0E1
ftsQ
K. Beggs
NK5012
supE,supF
N. Kleckner
CSR60 3
recAl,uvrA6,phr-1
B. Bachmann (CGSC #5830)
PoII1734
mini-MudII1734
M. Casadaban (20)
El 77
dnaA
B. Bachmann
AMC290
lacAX74
(23),TEY resistant,proC,trp
AMC419
ftsZ84::TnlO
A. Markovitz
AMC421
ftsZ+::TnlO
A. Markovitz
AMC413
ftsl(ts)::TnlO(leu)
A. Markovitz
YC99
sulB9::TnlO
This work
YC100
ftsAlO::TnlO(leu)
This work
YC200
lac+
lac+ derivative of AMC290
Hfr3000
supQ80,relAl,spoT,
B. Bachmann
82
Table 2.
Plasmids and phages.
Plasmids
Relevant markers
Source
PRGC31
ampr, ftsZ+,ftsA+
R. Gayda(23)
pRGC43
amp*",kan*",f tsA*
ftsZ::mini-MuI1734(lac*)
R. Gayda(23)
pGW7
amp1"
(30)
PKK232-8
amp^
J. Brosius(16)
pYCl6
kan^.ftsZ""
ftsA::mini-MuII1734(lac”)
This work(23)
V.anr ,ftsZ*
ftsA::mini-MuIII734(lac*)
This work(23)
kan1",ftsZ*
f tsA::mini-Mul117 34 (1aC")
This work(23)
kanr,ftsZ^
ftsA::mini-MulII734(lac*)
This work(23)
amp^.cm1amp^.cm1"
ampr,cmr
amp r,cm*"
amp^.cm^
This
This
This
This
This
pYC28
pYC39
pYC40
PYC500
PYC530
PYC580
pYC600
PYC620
work
work
work
work
work
Phages
PlVir
X1098
(125)
R. Gayda
X::mini-TnlO
N. Kleckner
84
Table 3.
Cellular Location of FtsA and FtsA-LacZ Fusions
Cellular Fractions
cytoplasmic*___________ total membranes1
Sarkosy
extracted fraction1
Plasmids
FtsA
P-gal fusion
pRG43
.14
.05
pRG31
.13
FtsA
.18
E-gal fusion
<.01
FtsA
.07
.12
E-gal fusion
<.01
.06
pYC40
(142,000 fusion)
.04
.05
.04
pYC28
(154,000 fusion)
.03
.04
.03
1 - Relative molar yield of plasmid encoded proteins.
Relative molar yield = % of polypeptide/molecular weight X 103
85
Table 4.
Plasmids
CAT/P-lactamase specific activities for different promoter
gene fusions.
Restriction frag.
fused to Cmr gene
Relevant
Promoters
PKK232-8
CAT/B-lactamase
activity
0
PYC600
BamHl - ficoRl
Pa.PzsPz 2 Pz 1
108
PYC620
Bglll - EcoiRl
Pz3Pz2P*l
59
PYC580
Hindlll-EcoRl
P2 2 PZ1
22
PYC530
Bglll-Hindlll
PZ3
235
pYC500
Bam\-Hindlll
Pa.Pz 3
249
Cell extracts of AMC290 containing above plasmids were prepared and
CAT specific activity/0-lactamase activity was determined as described
in Material and Methods. The background CAT activity in this assay was
zero in AMC290(0DSOo = 0.5). All measurements were performed in
duplicate and the mean is presented.
86
Table 5.
Plasmids
FtsZ effects on promoters.
Restriction frag.
fused to Cm*- gene
CAT/fl-lactamase
Relevant
Promoters
AMC419(ftsZ) AMC421(wt)
time 30°C 4*°C
30°C 43°i
(min)
PYC500
PYC530
BarrMl-Hindlll
Bglll-Hindlll
P*P23
Z3
30
314
458
256
819
60
400
583
nd
nd
30
164
377
244
930
60
179
507
nd
nd
nd = not done. A portion of actively growing cells (0D6Oo=0.1) were
temperature shifted to 43°C and the cells were harvested after 30 and 60
minutes as described in Material and Methods.
Table 6.
Plasmids
FtsQ effects on promoters.
Restriction frag.
fused to Cm*- gene
Relevant
Promoters
CAT/P-lactamase
activity X 103
TOEl(ftsQ)
time
30°C 43°C
(min)
PYC500
PYC530
BaciHl-ifindlll
Bglll-Hindlll
P^PZ3
P«
0
334
30
369
957
60
325
1184
0
331
30
319
709
60
219
1158
Cell extracts were prepared and the activity was assayed as described
in Table 5.
88
Table 7.
Plasmids
FtsA effects on promoters.
Restriction frag.
fused to Cm1- gene
Relevant
Promoters
CAT/P-lactamase
activity X 10s
YClOO(ftsAlO)
AMC290(wt)
time
30°C 4*°C 30°C
43°C
(min)______________________
PYC530
Bglll-Hindlll
Pz3
30
234
208
54
94
PYC580
Hindll-EcoBl
Pz2PZi
30
26
18
17
12
Cell were grown in L plus 0.2% glucose medium. Cell extracts were
prepared and the activity was assayed as Table 5.
Table 8.
Plasmids
DnaA46 mutants effects on promoters.
Restriction frag,
fused to Cmr gene
Relevant
Promoters
CAT/8-lactamase
activity X 103
E177(dnaAts)
time
30°C 43°C
(min)
PYC500
pYC530
PYC580
BarMl-Hindlll
Bglll-Hindlll
Hindlll-EcoRl
PaP z 3
^23
PzaPzi
0
114
60
131
0
84
60
88
0
8
60
9
201
186
11
Strains culture was grown overnight in LB-0.2% glucose at 30°C and
then diluted 100 fold and placed at 30°C. A portion of exponentially
growing cells (0D6Oo = 0.5) was temperature shifted to 43°C for 60
min. The cells were collected and extracts prepared for assays as
described in Materials and methods.
90
Fig. 1.
Locations of coding sequences and promoters within the ddl-
envA region of the E. coli chromosome,
(a) The 14 loci concerned with
cell envelope growth, organization and division have been identified
within the 2-min region of the E. coli chromosome.
enzyme map
of
structural
gnes.
transcription.
the
ddl-env.A region and,
P,
promoter
locations;
beneath,
(b) Restriction
location
arrows,
Abbreviations for restriction sites:
of
the
directions
of
B, BamHl;
Bg,
Bglll; C, Clal; E, £coRl; H, Hindlll; K, Kpnl; P, PstI; Pv, PvuII; S,
Sail; X, Xhol.
m r aA m ra B
B
ESSPX
ftsl tnurE m u r F tnurG m ur C ddl
ftsQ ftsA ftsZ envA secA azi
H
F. B K
rr
1L
pv
ddl
f tsQ
pw
pw
JT
p*
P* pi
f tsZ
f tsA
3
kilobases
J
I. i
envA
1
92
Fig. 2.
Proposed steps in the morphogenesis of E. coli cells.
morphogenic systems are represented.
of
enzymes
involed
in
the
net
Three
System i: gene specifying a set
synthesis
of
peptidoglycan(P.G.)
including mraA, mraB, mrhA, mrbB, mrbC, mrcA, mrcB, murE, murf, murG,
murC, ddl and possibly dacA and dacB.
System ii: shape modifying
system for the cylindrical P.G. sacculus.
gene products
assigned
formation,
for
(those
of
ftsZ,
ftsl,
successive
steps
in
completion,
separation
division sites) of new cell poles.
ftsQ,
cell
and
System iii: At least six
ftsA,
envA,
division;
inactivation
the
(as
and minB)
initiation,
potential
The phenotype of cells which have
been blocked at different stages in system iii division process are
shown at the top of the diagram.
OCDC
-> C D C D
CZXZD
ftsA, ftsl
1
mra A, B
mrb A,B,C
mrc A, B
mur E,F,G,C
ddl
(dac A ,B)
~r
(i)
)Q
envA
minB
94
Fig. 3.
Diagram of periseptal annuli in lkyD~ cells (a). Surface view
of the chain of cells (b).
Abbreviations: IM, inner membrane; M,
murein; OM, outer membrane; P, plasmolysis bays; D, zones of membranemurein adhesion that form the periseptal annuli; D', zones of adhesion
that form incomplete annuli at the mid points of the cells; S, septal
cleft.
(Modified from MacAlister et al (78)).
95
B
96
Fig. 4.
line,
Schematic representation of the expression vector pGW7. Thick
DNA
pBR322(30).
derived
from
phage
X;
thin
line,
DNA
derived
from
Arrows indicate the direction of transcription initiating
from the X Pt. and X PR promoters.
97
Amp
98
Fig. 5.
The vector pKK232-8.
A partial restriction map shows the
site in the polylinker used in promoter cloning.
Amp;P-lactamase gene,
T; terminators,
CAT; chloramphenicol gene. Restriction enzyme
codes: Ri, EcoRl; H3, Hindlll.
99
6
I
1---------- 1-----------1-----------1-----------f-
kb
Rl
pvull
Psti
H3 R l
Pvull
5.1 K b
txr— ►
806066 terminators
100
Fig. 6.
Restriction maps of pRGC31, Mudll 1734 and mini-Mu insertion
locations.
insertion
Encoding sequence for ftsA showed as hatch line.
sites
are
shown
plasmid designations.
by
lollipop
structure
with
Mini-Mu
associated
Restriction enzyme codes: Rl, EcoRl; pst, Pstl;
Bl, BamHl; H3, Hindlll.
Kanr is abbreviation for kanamycin resistance
(25
abbreviation
jig/ml).
pg/ml).
Ampr is
for ampicillin
resistance
(50
1k t
St
H----------A
E,
— I------------------------- H M u d
y
9 .7 Kb
i* c
Pst
li­
E ,
~l------- 1
ft S A
K1734
Z'
Kan
c,
6,
Amp'
pRGC3 i
7.8 K D
101
102
Fig. 7.
Autoradiograph of plasmid-specified polypeptides labelled in
UV-irradiated maxicells.
Polypeptides were separated on a 10% to 30%
gradient SDS-PAGE.
Lane A contained approximately 50% of the total
radioactivity
was
that
added
to
lanes
B,C,D
(500,000
c.p.m.).
Molecular weight standards used were: myosin (205 kD), P-galactosidase
(116 kD), phosphorylase B (97.4 kD), albumin, bovine (66 kD), albumin,
egg(45 kD), carbonic anhydrase (29 kD).
CSR603(pYC40);
FtsA-LacZ
Lanes: 1, CSR603(pRG31); 2,
3, CSR603(pYC28); 4, CSR603(pYC16).
fusions,
FtsA,
FtsZ,
protein specified by mini-Mu).
Bla(P-latamase,
Label point to
Amp*")
and Kan(Kanr
103
—CO
O
0
QC
Q.
o
CO
CM
CO
_
O
O o
> >• >
Q. Q. Q.
20511697-
66
^
‘- F t s A - L a c Z
-
45-
FtsA
«-FtsZ
♦kV*.
A B C
D
.104
Fig.
8.
gradient.
disrupted
Separation
of
membrane
fraction
Strain AMC290(pYC40)
and
(Section XV).
fractionated
Areas
as
by
sucrose
equilibrium
and AMC290(pRGC43)
described
corresponding
to
in materials
0MH , 0ML, F/B
were
and methods
and
described by Ishidate et al. (59) are indicated at top of Figure.
IM
as
105
OM
OM
F/ B
2. 0 - -
- ■
1.28
- -
1.24
- -
1.20
FtsA-LacZ
1.16
■1.12
1 .0 -
< La c Z
10
20
Fr a ct i o n Number
30
3
(g S u c r o s e / m l )
B-galactosidase
units/ml
3.0
106
Fig. 9.
pYC3.
Schematic diagram for construction of expression plasmid,
DNA segments of pYC16 (FtsA-LacZ plasmid) and vector pGW7 (X PL
promoter plasmid regulated by thermolabile Cl repressor , Cxs57) were
combined to generate pYC3.
ftsA
gene;
PL, K
PL
[
promoter;
], MudII1734 DNA (9.7 Kb); m h h b ,
kanr, kanamycin
resistance;
amp^,
ampicillin resistance; Z', B-galactosidase(truncated); N, X N gene.
Restriction enzyme codes: Rl, £coRl; P, flstl; Bl, BamHl; H3, Hindlll\
Bg, Belli.
107
BH
BH
pCW7
8. 0K b
857
p Y C 16
17.5Kb
f tsA
Kan
BH
Amp
Lac
BH j d i g e s t i o n
Bacterial
and
alkaline
phosphatase
treatment
Partial
digestion
BHj
purification
of
and
8.5Kb
DNA
Li ga t ion
Selection
BH
Be
857
p YC3
1 6 . 5Kb
Lac
E
Amp
BH
(Lac
+
, Amp
r
, Kan
5
)
with
fragment.
108
Fig. 10.
protein
SDS-PAGE profile of proteins for purification of FtsA-LacZ
from
plasmid pYC3.
temperature
shifted
AMC290(Alac)
strain
containing
The cells were shifted to 42°C for 2.5 hrs.
The hybrid
protein was purifed from the soluble fraction of French pressure lysed
cells by ammonium sulfate precipitation and affinity chromatography on
p-aminobenzyl-0-D-thio-galactopyranoside
in the Material and Methods.
agarose(Sigma)
as
described
Polypeptides were separated on a 10 to
30% gradient gel and stained with Coomassie blue (0.2% w/v).
A. molecular wight standards( same as described in Fig. 8);
protein in broken cells (140 yg);
(130 yg);
D.40%
Lanes:
B. total
C. total protein in cleared lysate
(Nm)2S0A pellet(130 yg);
E. purified FtsA-LacZ
fusion protein eluted from p-aminobenzyl-B-D-thio-galactoside column
(10 yg).
109
F tsA -L acZ
**
LacZ
110
Fig.
11.
protein
Identification of FtsA by Western blot analysis.
was
[35S]methionine
labelled
specifying FtsA and/or P-galactosidase.
min
at
100°C
in
mercaptoethanol.
sample
buffer
Proteins
were
electro-transferred
onto
Nytran
in
maxicells
with
FtsA
plasmids
Each sample was heated for 5
containing
4M
urea
separated
by
sheets.
Blocked
and
1%
SDS-PAGE(10-30%)
membranes
Pand
were
incubated with purified IgG antibodies (1:1000 dilution) against FtsALacZ for 2 hrs and washed.
Immunogenicity was detected with alkaline
phosphatase linked goat-anti-rabbit-IgG with color development by BCIP
and NBT.
Maxicell
labelled polypeptides were detected in the same
blot by autoradiography.
A).
Autoradiogram of blot B.
CSR603/pRGC43; lane 2 - CSR603/pRGC31.
of FtsA in total cells (blot B).
CSR603/pRGC31.
Lanel - CSR603/pRGC43; lane 2 Lane 1 - CSR603/pRGC31;
(1/5 cpm); lane3 - CSR603/pYC40
fusion); lane 4 - CSR603/pYC16 (FtsA-LacZ fusion).
detection of FtsA or FtsA-LacZ fusion (blotD).
pRGC31 specifies FtsA, FtsZ, Amp.
FtsA, FtsZ, Amp, Kan.
-
B). Immunoblotting detection
C). Autoradiogram of blot D.
lane 2 - CSR603/pRGC43
Lanel
(FtsA-LacZ
D). Immunoblotting
lanes 1-4 same as C.
pRGC43 specifies 3-galactosidase,
Ill
Fts A-Lac
FtsA
FtsA
112
Fig. 12.
Immunodetection of FtsA in AMC290 strain harboring pYC40 or
overproducing
performed
12.
plasmid
pYC3.
Western
blotting
experiments
were
under same conditions as described in the legend of Fig.
Lanes: A.
40% (NHA)2SOA precipitate of AMC290/pYC40 extract (150
yg); B. 40% (NHa )2S0* precipitate of temperature induce AMC290/pYC3
extract (65 yg).
113
A
B
114
Fig. 13.
Cellular localization of the FtsA protein by Western blot
with FtsA antibodies.
Membrane fractions of AMC290 were isolated by
the procedure of Ishidate et al. (59) as described in Fig. 9.
sample(~50yg)
was
heated
containing 4M urea and
for
20
min
at
90°C
1% P-mercaptoethanol
Western blot analysis.
in
sample
Each
buffer
prior to SDS-PAGE and
The Western blot was done under the same
experimental conditions described in Fig. 12.
Lanes: 1. IM - inner
membrane; 2. F/B - Flagellar fraction with membrane fragments; 3.
- outer membrane
light
fraction;
4.
0MH - outer membrane
0ML
heavy
fraction;5. Total membrane; 6. Total soluble proteins; 7. Control- 40%
(NHa )2S0*
of
temperature
induced
membrane; 9. Total soluble proteins.
AMC290/pYC3
extract;
8.
Total
U5
"'S
1 2
3
4
5
6
7
8
9
116
Fig. 14.
Western blot with FtsA-LacZ antibodies of cell fractions of
AMC290.
Approximately equal amount
fractions
of
AMC290
conditions were
were
applied
as described
(~50yg) protein from cellular
to
in Fig.
each
14.
lane.
Lanes:
Experimental
A.
IM - inner
membrane; B. F/B - flagellar fraction with membrane fragments; C. OMt,
-
outer
membrane
fraction;
control
E.
-
light
fraction;
D.
total membrane pellet;
CSR603/pRGC31
total
cell
0M„
F.
-
outer
membrane
heavy
total soluble proteins;
proteins;
H.
(NH^)2S0* of temperature induced AMC290/pYC3 exract.
control
-
G.
40%
117
*K**tn.
*-A*fl%
A B C
D
E
F
G
H
FtsA
118
Fig. 15.
SDS-PAGE Coomassie blue stain gel profile of AMC290 cellular
fractions analysed in immunoblot of Fig. 15.
and separated as descibed in Fig. 15.
Each sample was prepared
Lanes: A. IM - inner membrane;
B. F/B - flagellar fraction with membrane fragments; C. OMr. - outer
membrane light fraction; D. 0M„ - outer membrane heavy fraction; E.
total
membrane
CSR603/pRGC31
pellet;
total
F.
total
cell proteins;
soluble
H.
temperature induced AMC290/pYC3 exract.
proteins;
control
- 40%
G.
control
-
(NHA)2S0* of
119
| I g _ J |||
66
-
mum.- m*ms*
45 -
29-
Vjfl
1 2
3
4
5 6 7 8 9
120
Fig.
16.
Western
blot of
FtsA antibodies
membrane of cell fractions of AMC290.
preabsored with
total
Approximately equal volume of
each sample was prepared and analyzed as described in Fig. 15.
FtsA-
LacZ specific antibody faction was preabsorbed twice with lysate of
ftsAlO mutant containing the total membrane fractions isolated from a
sucrose step gradient. Lanes: 1. IM - inner membrane(60 yg); 2. F/B flagellar fraction with membrane
membrane
light
fraction(60
yg);
fragments(10 yg);
4.
0M„
-
outer
3. 0ML - outer
membrane
heavy
fraction(40 yg); 5. total membrane pellet(50 yg); 6. total soluble
proteins(50 yg); 7. control - CSR603/pRGC31 total cell proteins; 8.
control - 40% (NHa)2SOa of temperature induced AMC290/pYC3 exract(50
yg).
121
'r. v:"^ /i-
• . i
Ft s A
:
8111#
?■••• 'l\ .■;.;
'■=M
2
3
~
4
5
6
7
8
122
Fig. 17.
Electron microscopic localization of FtsA in E. coli cells
by immunogold labelling. Cells were embedded, thin sectioned, treated
with FtsA-LacZ polyclonal
rabbit
IgG
colloidal
Material and Methods.
antibodies
gold
and
labelled with goat
conjugates(lOnm)
as
described
Lanes: A,B,C,D - AMC290(Alac)
in
anti­
the
treated with
FtsA-LacZ polyclonal antibodies; E,F - control, AMC290 treated with
pre-immune serum antibodies; G,H - control, YC200(lac*) treated wtih
FtsA-LacZ polyclonal antibodies.
123
•Mt
,»■
r
\y&uF^
^0T
w
••' »r..
.
124
125
Fig. 18.
Phase contrast light and electron micrographs of plasmolyzed
ftsZ84(ts) filaments.
Strain AMC419 growing cells at 30°C (0DfeOo =
0.2) in YET broth were shifted to 42°C for 90 min.
A portion of the
culture was plasmolyzed in 13% sucrose, fixed with paraformaldehyde
and
prepared
for
phase
contrast
(A)
and
electron
microscopy(B).
Plasmolysis bays are visible along the lenght of the filaments.
pm - light micrograph; Bar,
! pm
- electron micrograph.
Bar,
126
A
127
Fig. 19.
Immunoelectron micrographic localization of FtsA in ftsZ8A
filament.
A
portion
paraformaldehyde
and
of
culture
prepared
described in the legend to Fig. 18.
in
for
Bar,
Fig.
18
was
fixed
immuno-electromicroscopy
| ym.
with
as
128
129
Fig.
20.
Localization of FtsA in plasmolyzed ftsZ84 filament by
immunoelectron
micrograph.
This
section
of
plasmolyzed
ftsZ
filamentous cells in Fig.
18 was subject ot immunogold labelling as
described in Fig. 18.
} pm.
Bar
130
131
Fig. 21.
Promoter fusions of the ftsA-ftsZ regulatory region to the
chloramphenicol acetylase gene(Cm*").
A partial restriction map of the
2.2 Kb fragment from pRGC31(Fig.6 ) is shown together with the extent
of cloned fragments and their orientation relative to Cm*" structural
gene in pKK232-8.
and the structural
Also depicted are the promoter (Pzl, Pz2, Pz3, PA )
regions of ftsQ,
incomplete coding region).
ftsA,
ftsZ.
(" ' " indicated
Abbreviation used for restriction enzymes
are Ei, EcoRl; Bi, BamHl; Bg, Bglll; Bs, BssHI; H3, Hindlll.
*t»Q'
UtZ‘
«HA
BomHi
BoMi
fc= r-
Hindiu
-i
EcoRl
1 i,l
- >
—
PA
—
>
PZ3
»
PZ21
^ H3
pYC 500
Z3
Bg
pYC 530
■>H:
Z3
H,
pYC 580
Z21
r > El
PZ3
PZ2l
B g ----------P
^
Z3
pYC 600
P
Z21
pYC 620
VITA
Younghae Chong Chon was born August 4, 1953 in Seoul, Korea.
She graduated from Jeongshin Girls High School in Seoul, Korea in
1972.
She attended
Ewha Women's
University
received a Bachelor of Pharmacy degree in 1976.
worked for more
Investigation
in
than 3 years at National
Seoul,
Korea
in
a
in Seoul,
Korea
and
After graduation she
Institute of Scientific
laboratory
as
a
pharmacist.
Younghae entered the Graduate School of Louisiana State University in
August 1982 and received the Master of Science degree in Microbiology
in December 1984.
She is currently a candidate for the degree of
Doctor of Philosophy in Microbiology at Louisiana State University.
133
DOCTORAL EXAMINATION AND DISSERTATION REPORT
Candidate:
Major Field:
Younghae Chong Chon
Microbiology
Title of Dissertation:
Cell Division Studies of Escherichia c o li ; Expression
and Protein Localization of a Cell Septation Gene, ftsA.
Approved:
Major Professor and Chairman
Dean of the GraduatfKschooI
EXAMINING COMMITTEE:
Date of Examination:
Tuesday, July 12, 1988